Methods of differentiating and protecting cells by modulating the P38/MEF2 pathway

ABSTRACT

The present invention provides a method of differentiating progenitor cells to produce a population containing protected neuronal cells. A method of the invention includes the steps of contacting the progenitor cells with a differentiating agent; and introducing into the progenitor cells a nucleic acid molecule encoding a MEF2 polypeptide or an active fragment thereof, thereby differentiating the progenitor cells to produce a population containing protected neuronal cells. In one embodiment, the MEF2 polypeptide is human MEF2C or an active fragment thereof.

[0001] This application is based on, and claims the benefit of, U.S.Provisional Application No. 60/209,539, filed Jun. 5, 2000, and which isincorporated herein by reference.

[0002] This application was made with government support under P01HD29587 awarded by the National Institute of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The invention relates to neuronal cell transplantation forneurodegenerative conditions of the central nervous system includinghypoxia-ischemia (stroke), Parkinson's disease, Huntington's disease,amyotrophic lateral sclerosis, Alzheimer's disease and other forms ofdementia and, more specifically, to methods of producing populations ofneurons by manipulating the myocyte enhancer factor 2 (MEF2)transcription pathway.

[0005] 2. Background Information

[0006] For a variety of serious neurodegenerative diseases, there existno effective therapies or cures. For example, Parkinson's disease is aprogressive and ultimately fatal neurodegenerative disordercharacterized by loss of the pigmented dopaminergic neurons of thesubstantia nigra. The symptoms of Parkinson's disease can often bemanaged initially by administration of L-DOPA, the immediate precursorof dopamine. However, reduced efficacy of L-DOPA treatment typicallyoccurs over time. Programmed cell death (apoptosis) has been implicatedin this neurodegenerative disorder.

[0007] In Alzheimer's disease, the most common neurodegenerative diseaseand most frequent cause of dementia, progressive failure of memory anddegeneration of temporal and parietal association cortex result inspeech impairment and loss of coordination, and, in some cases,emotionally disturbance. Alzheimer's disease generally progresses overmany years, with patients gradually becoming immobile, emaciated andsusceptible to pneumonia.

[0008] The brain constitutes a privileged transplantation site and,under the appropriate conditions, neuronal tissues can survivetransplantation into the damaged brain, integrate with the host andalleviate functional impairments associated with neurological disease.Neuronal cell transplantation has been sought for a variety of seriousneurodegenerative diseases for which no effective therapeutic courseexists, including Parkinson's disease and Alzheimer's disease as well asHuntington's disease, amyotrophic lateral sclerosis, multiple sclerosis,epilepsy and pain.

[0009] Present techniques for neuronal transplantation have chieflyrelied on embryonic or fetal tissues since central nervous system (CNS)neurons only survive transplantation if taken from embryonic or neonataldonors. Neuronal transplantation has been hampered by extremely limitedsupplies of human embryonic or fetal tissue. In order to developalternative supplies of donor neurons, scientists have attempted thelarge scale expansion of stem cells and precursor cells. When treatedwith high doses of epidermal growth factor, stem and precursor cellsfrom the brain can be selectively expanded in vitro and grownexponentially through multiple passages. These expanded cells, which canbe produced from human tissues, yield both neuronal and glial cell typeswhen allowed to differentiate in vitro and survive transplantation backinto animal central nervous system (CNS; Svendsen et al., Exp. Neurol.140:1-13 (1996)).

[0010] Unfortunately, the expansion of stem and precursor cellpopulations currently does not produce a cell population useful fortherapeutic transplantation, since a relatively small number of neuronsis produced, and even a smaller number survive and express the neuronalphenotype when grafted into the central nervous system. Thus, there is aneed for a method of efficiently producing large numbers of neuronalcells or their precursors which are capable of surviving whentransplanted into the central nervous system in vivo. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

[0011] The present invention provides a method of differentiatingprogenitor cells by contacting the progenitor cells with adifferentiating agent; and introducing into the progenitor cells anucleic acid molecule encoding a MEF2 polypeptide or an active fragmentthereof, thereby differentiating the progenitor cells to produce a cellpopulation containing protected neuronal cells. In one embodiment, theproduced population containing protected neuronal cells contains atleast 50% neuronal cells.

[0012] A method of the invention can be practiced, for example, with anucleic acid molecule encoding human MEF2C, or an active fragmentthereof. In one embodiment, the MEF2 polypeptide is constitutivelyactive. In another embodiment, the constitutively active MEF2polypeptide is a MEF2/VP16 fusion protein. In a further embodiment, theconstitutively active MEF2 polypeptide contains one or moreserine/threonine to aspartic acid/glutamic acid substitutions in theMEF2 transactivation domain.

[0013] A method of the invention for differentiating progenitor cells toproduce a cell population containing protected neuronal cells canfurther include the step of inhibiting caspase activity in theprogenitor cells.

[0014] Progenitor cells useful in the methods of the invention can be,for example, human stem cells. In one embodiment, the progenitor cellsare embryonic stem cells, for example, human embryonic stem cells. Inanother embodiment, the progenitor cells are hematopoietic progenitorcells, for example, human hematopoietic progenitor cells.

[0015] In one embodiment, a method of the invention further includes thestep of selecting CD133-positive (CD133-positive) human progenitorcells. In another embodiment, a method of the invention includes thestep of selecting CD133-positive/CD34-positive human progenitor cells.In a further embodiment, a method of the invention further includes thestep of selecting CD133-positive/ CD34-negative human progenitor cells.In yet further embodiments, CD133-positive/CD34-negative/CD45-negative,or CD34-negative/CD38-negative/Lin-negative human progenitor cells orCD34-positive/CD38-negative/Lin-negative/Thy-1-negative human progenitorcells are selected.

[0016] For use in a method of the invention, the differentiating agentcan be, for example, retinoic acid. Other differentiating agents usefulin a method of the invention for producing a cell population containingprotecting neurotrophic factor 3, epidermal growth factor, insulin-likegrowth factor 1 and a platelet-derived growth factor.

[0017] In a further embodiment, a method of the invention furtherincludes the step of transplanting cells containing a nucleic acidmolecule encoding a MEF2 polypeptide or an active fragment thereof intoa patient to produce a cell population containing protected neuronalcells in the patient.

[0018] The invention further provides an isolated stem cell thatcontains an exogenous nucleic acid molecule encoding a MEF2 polypeptideor an active fragment thereof. The isolated stem cell can include, forexample, a nucleic acid molecule encoding a MEF2 polypeptide, or activefragment thereof, operatively linked to a heterologous regulatoryelement. The encoded MEF2 polypeptide can be, for example, a human MEF2polypeptide. If desired, the encoded MEF2 polypeptide can be a MEF2Cpolypeptide. In a further embodiment, the MEF2 polypeptide isconstitutively active. Such a constitutively active MEF2 polypeptide canbe, for example, a constitutively active MEF2C polypeptide. In oneembodiment, the constitutively active MEF2 polypeptide is a MEF2/VP16fusion protein. In another embodiment, the constitutively active MEF2polypeptide contains one or more serine/threonine to asparticacid/glutamic acid substitutions in the MEF2 transactivation domain.

[0019] An isolated stem cell of the invention can be, for example, ahuman stem cell. In one embodiment, the stem cell is an embryonic stemcell, for example, a human embryonic stem cell. A human stem cell of theinvention can contain, for example, an exogenous nucleic acid moleculeencoding human MEF2C. A human stem cell of the invention also caninclude a constitutively active MEF2 polypeptide.

[0020] The invention further provides an isolated hematopoietic stemcell that contains an exogenous nucleic acid molecule encoding a MEF2polypeptide or an active fragment thereof. Such an isolatedhematopoietic stem cell can include, for example, a nucleic acidmolecule encoding a MEF2 polypeptide, or active fragment thereof,operatively linked to a heterologous regulatory element. In oneembodiment, an isolated hematopoietic stem cell of the invention is ahuman hematopoietic stem cell.

[0021] The present invention also provides a method of identifying aprotective or differentiation gene, which can be, for example, aneuroprotective gene or a gene that contributes to neuronal or musclecell differentiation. A method of the invention includes the steps ofisolating a first cell population; isolating a second cell population,wherein the second cell population has an altered level or activity of aMEF2 polypeptide as compared to the first cell population; and assayingfor differential gene expression in the first cell population ascompared to the second cell population, whereby a gene differentiallyexpressed in the second cell population as compared to the first cellpopulation is identified as a protective or differentiation gene. In oneembodiment, the first cell population is a progenitor cell population,the second cell population is a neuronal cell population, and thedifferentially expressed gene is a neuronal differentiation gene. In afurther embodiment, the first cell population is a progenitor cellpopulation, the second cell population is a muscle cell population, andthe differentially expressed gene is a muscle differentiation gene. Inyet another embodiment, both cell populations are neuronal cellpopulations, the second cell population has been subject to a neuronalstress as compared to the first cell population, and the differentiallyexpressed gene is a neuroprotective gene.

[0022] Further provided by the invention is a method of identifying aprotective gene in vitro. The method is practiced by inducing thep38/MEF2 pathway in a cell in vitro to produce a protected cell;stressing the cell; and assaying for differential gene expression in theprotected cell as compared to gene expression in a control cell, wherebya gene differentially expressed in the protected cell as compared to thecontrol cell is identified as a protective gene. In such a method of theinvention, the p38/MEF2 pathway can be induced, for example, byintroducing into the cell a nucleic acid molecule encoding a MEF2polypeptide. The MEF2 polypeptide can be, for example, a human MEF2polypeptide and further can be, if desired, a constitutively active MEF2polypeptide. In one embodiment, a neuroprotective gene is identified byinducing the p38/MEF2 pathway in a neuron. In another embodiment, amuscle protective gene is identified by inducing the p38/MEF2 pathway ina muscle cell. In a method of the invention, the differential geneexpression that identifies the protective gene can be increased ordecreased gene expression.

[0023] The invention additionally provides a method of identifying adifferentiation gene in vitro by inducing the p38/MEF2 pathway in aprogenitor cell in vitro to produce a differentiated cell; and assayingfor differential gene expression in the differentiated cell as comparedto gene expression in a control cell, whereby a gene differentiallyexpressed in the differentiated cell as compared to the control cell isidentified as a differentiation gene. In a method of the invention, thep38/MEF2 pathway can be induced, for example, by introducing into theprogenitor cell a nucleic acid molecule encoding a MEF2 polypeptide. TheMEF2 polypeptide can be, for example, a human MEF2 polypeptide or aconstitutively active MEF2 polypeptide. In one embodiment, thedifferentiated cell is a neuronal cell, and, in a further embodiment,the differentiated cell is a muscle cell. The differential geneexpression which serves to identify the differentiation gene can beincreased or decreased gene expression.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1A shows schematic diagrams of MEF2 factors. Structures ofthe four vertebrate mef2 gene products are shown. Alternative exonswithin the C-terminal activation domains are indicated, along with thenumber of amino acids in the longer form of each protein. FIG. 1B showsconserved regions in the C terminus of MEF2 contain potentialphosphorylation sites. MEF2A motif 1 to 4 are shown as SEQ ID NOS:13 to16, respectively. MEF2C motif 1 to 4 are shown as SEQ ID NOS:17 to 20,respectively. MEF2D motif 1 to 3 are shown as SEQ ID NOS:21 to 23,respectively. Sites that are phosphorylated by p38and ERK5 are markedwith asterisks. Not all potential phosphorylation sites are shown. Someof the conserved stretches overlap with transactivation domains thathave been mapped by deletion analysis. PKC, protein kinase C site: MAPK,mitogen-activated protein kinase site; CKII casein kinase II site.CaMKIV, calcium-calmodulin kinase IV site. MEF2A motif 1 to 4 are shownas SEQ ID NOS:13 to 16, respectively. MEF2C motif 1 to 4 are shown asSEQ ID NOS:17 to 20, respectively. MEF2D motif 1 to 3 are shown as SEQID NOS:21 to 23, respectively.

[0025]FIG. 2A shows the nucleotide sequence (SEQ ID NO:1) of human MEF2A(GenBank accession NM 005587). FIG. 2B shows the amino acid sequence(SEQ ID NO:2) of human MEF2A.

[0026]FIG. 3A shows the nucleotide sequence (SEQ ID NO:3) of human MEF2B(GenBank accession NM 005919). FIG. 3B shows the amino acid sequence(SEQ ID NO:4) of human MEF2B.

[0027]FIG. 4A shows the nucleotide sequence (SEQ ID NO:5) of human MEF2C(GenBank accession L08895). FIG. 4B shows the amino acid sequence (SEQID NO:6) of human MEF2C. The MADS domain is bolded while the MEF2 domainis underlined.

[0028]FIG. 5A shows the nucleotide sequence (SEQ ID NO:7) of human MEF2D(GenBank accession NM 005920). FIG. 5B shows the amino acid sequence(SEQ ID NO:8) of human MEF2D.

[0029]FIG. 6 shows MEF2 binding activity, protein expression andtransfection during neuronal differentiation of P19 stem cells. (A) Gelshift assays show that MEF2 binding activity increased during neuronaldifferentiation of P19 cells. A ³²P-labeled MEF2 site oligonucleotidewas incubated with nuclear extracts from undifferentiated P19 cells(lanes 1 and 3), or from P19 cells treated with retinoic acid for 2 d(lanes 2 and 4). Cold competition by unlabeled MEF2 siteoligonucleotides (lanes 3 and 4). (B) Antibody to MEF2A (lane 6), MEF2C(lane 7), or MEF2D (lane 8) was added to the binding mixture forsupershift assays. Anti-MEF2C yielded two supershifted bands,representing one or more DNA complexes containing MEF2C, whileanti-MEF2D produced a single supershifted complex (arrows; Leifer etal., Proc. Natl. Acad. Sci. USA 90:1546-1550 (1993)). (C) Immunoblotsrevealed that protein expression of MEF2C and MEF2D was induced duringneuronal differentiation of P19 cells. Whole cell lysates fromundifferentiated P19 cells or P19 cells treated with retinoic acid for 2days were used for these immunoblots (n.s., non-specific bands). (D-G)Overexpression of MEF2C induced a mixed neurogenic/myogenic phenotype.Undifferentiated P19 cells did not display immunoreactivity for MEF2C orneurofilament (D, phase contrast image; E, immunocytochemistry).Undifferentiated P19 cells were transfected with an expression vectorfor MEF2C. Similar findings were observed in 16 experiments in whichover 200 cells were scored.

[0030]FIG. 7 shows inhibition of MEF2 function decreases the number ofneuronal (MAP2-positive) P19 cells after retinoic acid treatment. (A)Undifferentiated P19 cells were stably transfected with empty vector(clones 2-1, 2-2 and 2-5 in lanes 1-3, respectively) or MEF2 dominantnegative (clones 2-7, 2-8 and 2-9 in lanes 4-6, respectively).Expression of the MEF2 dominant negative was demonstrated in a gel shiftassay using a radiolabeled MEF2 site oligonucleotide and nuclearextracts of each clone. DN: binding complex of the MEF2 site anddominant negative MEF2 protein. (B and C) Cultures from control (B,clone 2-1) and MEF2 dominant negative (C, clone 2-7) transformants weretreated with retinoic acid for 7 d to induce neurogenesis, and neuronaldifferentiation was then evaluated by immunocytochemistry withanti-MAP2. (D) Control cultures (clones 2-1, 2-2, 2-5) and MEF2 dominantnegative cultures (clones 2-7, 2-8, 2-9) were treated with retinoic acidand scored for the number of MAP2-positive cells (n=6 experiments; *,P<0.0001 by ANOVA and post-hoc comparison).

[0031]FIG. 8 shows that inhibition of MEF2 function decreases the numberof multipotent and unipotent precursor cells. Control cultures (clone2-1) and MEF2 dominant negative cultures (clone 2-7) were treated withretinoic acid for 3.0 d or 3.5 days. (A and C) Cells incubated withanti-nestin to label multipotent precursor cells (A) or anti-Hu to labelunipotent precursor cells (C). Labeled cells were visualized withperoxidase. (B and D) The number of nestin-positive cells (B) andHu-positive cells (D) in 40 randomly selected fields was scored in ablinded fashion. Values are mean ±SD from at least three independentexperiments (*, P<0.02 by Student's t-test).

[0032]FIG. 9 shows the effects of inhibition of MEF2 function duringneuronal differentiation of P19 cells. Control cultures (clone 2-1),MEF2 dominant negative cultures (clone 2-7, labeled DN) and mutated MEF2dominant negative cultures (clone 2-16, labeled DNmt) were treated withretinoic acid for 3 days. (A) Representative apoptotic cells withcondensed nuclei from a MEF2 dominant negative clone treated withretinoic acid and stained with Hoechst dye to detect apoptoticmorphology (white arrows). (B) Percentage of apoptotic cells in controlor MEF2 dominant negative cultures before and after retinoic acidtreatment. (C) Similar percentage of apoptotic cells in control ormutated MEF2 dominant negative cultures after 3 days of retinoic acid.(D) Apoptosis in control, dominant negative or mutated dominant negativecultures treated with retinoic acid for 3 days scored by the TUNELtechnique. (E and F) Lack of effect of MEF2 dominant negative onmultipotent precursor cell proliferation. Control cultures and MEF2dominant negative cultures were treated with retinoic acid for 3 days.BrdU was then added to visualize proliferating cells. (E) Dividingmultipotent precursor cells detected by double staining with anti-BrdUantibody and anti-nestin antibody in retinoic acid-treated controlcells. (F) Comparison of BrdU incorporation into multipotent(nestin-positive) precursor cells in control and MEF2 dominant negativecultures. Values are mean ±SD from at least three independentexperiments (*, P<0.05 by Student's t-test; t, P<0.001 by ANOVA andpost-hoc comparison).

[0033]FIG. 10 shows involvement of the p38α(/MEF2 pathway in preventingapoptosis during neuronal differentiation of P19 cells. (A) p38α wasphosphorylated during induction of neuronal differentiation by retinoicacid. Anti-phospho p38 was used to detect activated/phosphorylated p38family members on immunoblots during induction of neuronaldifferentiation. The same membrane was then stripped and re-blotted witha p38α-specific antibody that labeled the same band. (B) Dominantnegative p38α (p38αDN) enhanced apoptosis during neuronaldifferentiation. Constitutively active MEF2C (MEF2C/VP16) significantlyrescued the differentiating cells from apoptosis. Dominant negativep38β₂ (p38b₂DN) had no effect on apoptosis compared to control(expression vector only). After treatment with retinoic acid for oneday, cells were transfected with the indicated expression vector(s)along with a GFP expression vector to identify the transfected cells.The number of transfected apoptotic cells was determined in a blindedfashion by TUNEL assay on day 3 of retinoic acid treatment. Over 1200GFP-positive cells were scored in each culture. Mean±SD are shown fromthree experiments P(*, P<0.001; †, P<0.01 by ANOVA and post-hoccomparison).

DETAILED DESCRIPTION OF THE INVENTION

[0034] MEF2 proteins are members of the MADS family of transcriptionfactors (Treisman, R., Nature 376:468-469 (1995)). Four members of thefamily (MEF2A, MEF2B, MEF2C and MEF2D) have been reported, includingmurine and human polypeptides (Pollock and Treisman, Genes & Dev.5:2327-2341 (1991); Leifer et al., Proc. Natl. Acad. Sci. USA90:1546-1550 (1993); Martin et al., Proc. Natl. Acad. Sci. USA90:5282-5286 (1993); Martin et al., Mol. Cell. Biol. 14:1647-1656(1994); and Molkentin et al., Mol. Cell. Biol. 16:3814-3824 (1996)), andone MEF2 homolog, D-MEF2, has been identified in Drosophila (Lilly etal., Proc. Natl. Acad. Sci., USA 91:5662-5666 (1994)). Various MAPkinases (p38α, p38β₂, and BMK1/ERK5) phosphorylate and thereby activateMEF2 family members (Han et al., Nature 386:296-299 (1997); Kato et al.,EMBO J. 16:7054-7066 (1997); Zhao et al., Mol. Cell. Biol. 19:21-30(1999); and Yang et al., Mol. Cell. Biol. 19:4028-4038 (1999)). TheseMAP kinase pathways lead to MEF2 modulation of gene expression (Han etal., Nature 386:296-299 (1997); Kato et al., EMBO J. 16:7054-7066(1997); and Zhao et al., Mol. Cell. Biol. 19:21-30 (1999)).

[0035] The MEF2 family of genes is highly expressed in cells of musclelineage, and several studies support a role for MEF2 in myogenesis(Martin et al., Proc. Natl. Acad. Sci. USA 90:5282-5286 (1993); Martinet al., Mol. Cell. Biol. 14:1647-1656 (1994); Molkentin et al., Mol.Cell. Biol. 16:3814-3824 (1996); and McDermott et al., Mol. Cell. Biol.13:2564-2577 (1993)). In D-mef2 loss-of-function Drosophila, musclecells have lost the ability to differentiate (Lilly et al., Science267:688-693 (1995)), and MEF2C-null mice are embryonic lethal due tomalformation of the heart (Lin et al., Science 276:1404-1407 (1997)).Furthermore, a dominant negative form of MEF2 also inhibits myotubeformation in myoblastic cell lines (Ornatsky et al., J. Biol. Chem.272:33271-33278 (1997)). During myogenesis, evidence indicates that MEF2proteins physically interact with the basic helix-loop-helix (bHLH)myogenic transcription factors MyoD and myogenin to initiate muscledevelopment (Kaushal et al., Science 266:1236-1240 (1994) and Molkentinet al., Cell 83:1125-1136 (1995)).

[0036] MEF2 family members including MEF2C also are highly expressed inneurons in the central nervous system (CNS) (Lyons et al., J. Neurosci.15:5727-5738 (1995); Leifer et al., Proc. Natl. Acad. Sci. USA90:1546-1550 (1993)). The level of MEF2 expression increases indifferentiating neurons in the developing brain (Leifer et al., Proc.Natl. Acad. Sci. USA 90:1546-1550 (1993); Lyons et al., J. Neurosci.15:5727-5738 (1995); Leifer et al., Neuroscience 63:1067-1079 (1994);and Lin et al., Mol. Brain. Res. 42:307-316 (1996)). Several neuronalbHLH transcription factors have been identified during mammaliandevelopment (Lee et al., Mol. Cell. Biol. 17:2745-2755 (1997)). Ectopicoverexpression of neuronal bHLH factors NeuroD(1)/BETA2,NeuroD2/KW8/NDRF, or NeuroD3/neurogeninl in Xenopus causes neurogenicconversion of ectoderm (Lee et al., Mol. Cell. Biol. 17:2745-2755(1997)). In addition, physical and functional interaction between aneuronal bHLH transcription factor (Mash-1) and MEF2 proteins has beenreported (Skerjanc and Wilton, FEBS Letters 472:53-56 (2000); Mao andNadal-Ginard, J. Biol. Chem. 271:14371-14375 (1996); and Black et al.,J. Biol. Chem. 271:26659-26663 (1996)).

[0037] The present invention relates to the finding that the p38α/MEF2pathway plays an important role in preventing apoptotic cell deathduring neuronal differentiation. Based on this finding, the presentinvention provides a method of differentiating progenitor cells toproduce a population of neuronal cells which is protected from apoptoticcell death. The method includes the steps of contacting the progenitorcells with a differentiating agent and inducing the p38mitogen-activated protein kinase/myocyte enhancer factor 2 (p38 MAPkinase/MEF2) pathway in the progenitor cells, thereby differentiatingthe progenitor cells to produce a population containing protectedneuronal cells. In one embodiment, the MAP kinase/MEF2 pathway isinduced by introducing into the progenitor cells a nucleic acid moleculeencoding a MEF2 polypeptide or an active fragment thereof. A MEF2polypeptide useful in a method of the invention can be, for example, ahuman MEF2 polypeptide or an active fragment thereof and, in oneembodiment, is a MEF2C polypeptide or active fragment thereof, forexample, a human MEF2C polypeptide or active fragment thereof.

[0038] In another embodiment, a method of the invention fordifferentiating progenitor cells is practiced by introducing into theprogenitor cells a nucleic acid molecule encoding a constitutivelyactive MEF2 polypeptide such as a constitutively active form of MEF2A,MEF2B, MEF2C or MEF2D. In a further embodiment, the constitutivelyactive form of the MEF2 polypeptide is resistant to caspase cleavage. Aconstitutively active MEF2 polypeptide useful in the invention can be,for example, a chimera in which the native MEF2 activation domain isreplaced with a heterologous activation domain, for example, aconstitutively active MEF2A/VP16, MEF2A/GAL4, MEF2B/VP16, MEF2B/GAL4,MEF2C/VP16, MEF2C/GAL4, MEF2D/VP16 or MEF2D/GAL4 fusion protein. Aconstitutively active MEF2 polypeptide useful in the invention also canbe, for example, a modified MEF2 polypeptide in which one or all of thep38 kinase phosphorylation sites in the MEF2 transactivation domain aresubstituted with an aspartic or glutamic acid residue. In a furtherembodiment, a method of the invention is practiced by introducing intothe progenitor cells a MEF2 activator, whereby the p38/MEF2 pathway isinduced. Such a MEF2 activator can be, for example, a nucleic acidmolecule encoding p38α.

[0039] In another embodiment, a method of the invention fordifferentiating progenitor cells to produce a population containingprotected neuronal cells is practiced by introducing into the progenitorcells a caspase inhibitor in addition to administering the agent thatinduces the p38/MEF2 pathway. It is understood that the caspaseinhibitor can be administered together with, prior to or followingadministration of the agent that induces the p38/MEF2 pathway. In oneembodiment, a method of the invention is practiced by introducing intothe progenitor cells a caspase inhibitor in addition to a constitutivelyactive MEF2 polypeptide.

[0040] In a further embodiment, the MAP kinase/MEF2 pathway is inducedby introducing into the progenitor cells a MEF2 activator such as anucleic acid molecule encoding p38α. A population containing protectedneuronal cells produced by a method of the invention can be made up of,for example, at least 50% neuronal cells. If desired, cells containing anucleic acid molecule encoding a MEF2 polypeptide or an active fragmentthereof can be transplanted into a patient, for example, into the brain,central nervous system or retina, to produce a cell populationcontaining protected neuronal cells in the patient.

[0041] The present invention also provides a method of reducing theseverity of a neurologic condition in a subject by administering to thesubject an agent that induces the p38 mitogen-activated proteinkinase/myocyte enhancer factor 2 (MEF2) pathway. A method of theinvention can be useful, for example, in reducing the severity of anacute neurologic condition such as cerebral ischemia; stroke; hypoxia;anoxia; poisoning by carbon monoxide, manganese or cyanide;hypoglycemia; mechanical trauma to the nervous system such as trauma tothe head or spinal cord; or epileptic seizure. A method of the inventionfurther can be useful, for example, for reducing the severity of achronic neurodegenerative disease such as Huntington's disease; adisorder of photoreceptor degeneration such as retinitis pigmentosa;acquired immunodeficiency syndrome (AIDS) dementia complex; aneuropathic pain syndrome such as causalgia or a painful peripheralneuropathy; olivopontocerebellar atrophy; Parkinsonism; amyotrophiclateral sclerosis; a mitochondrial abnormality or other biochemicaldisorder such as MELAS syndrome, MERRF, Leber's disease, Wernicke'sencephalopathy, Rett syndrome, homocysteinuria, hyperhomocysteinemia,hyperprolinemia, nonketotic hyperglycinemia, hydroxybutyricaminoaciduria, sulfite oxidase deficiency, combined systems disease,lead encephalopathy; Alzheimer's disease, hepatic encephalopathy,Tourette's syndrome, or drug addiction, tolerance or dependency. Thus,in one embodiment, a method of the invention reduces the severity ofstroke; hypoglycemia; trauma; epilepsy; neuropathic pain; peripheralneuropathy, for example, associated with diabetes mellitus; glaucoma;multiple sclerosis. In another embodiment, a method of the inventionreduces the severity of Alzheimer's disease, Huntington's disease,acquired AIDS dementia complex, or amyotrophic lateral sclerosis. In afurther embodiment, a method of the invention reduces the severity ofdepression, anxiety, or drug dependency, drug withdrawal or drugaddiction.

[0042] A method of the invention for reducing the severity of aneurologic condition can be practiced, for example, by administering toa subject a nucleic acid molecule encoding a MEF2 polypeptide or anactive fragment thereof. A MEF2 polypeptide useful in a method of theinvention can be, for example, a human MEF2 polypeptide or an activefragment thereof and, in one embodiment, is a MEF2C polypeptide oractive fragment thereof, for example, a human MEF2C polypeptide oractive fragment thereof.

[0043] In another embodiment, a method of the invention for reducing theseverity of a neurologic condition is practiced by administering to asubject a nucleic acid molecule encoding a constitutively active MEF2polypeptide such as a constitutively active form of MEF2A, MEF2B, MEF2Cor MEF2D. In a further embodiment, the constitutively active form of theMEF2 polypeptide is resistant to caspase cleavage. A constitutivelyactive MEF2 polypeptide useful in the invention can be, for example, achimera in which the native MEF2 activation domain is replaced with aheterologous activation domain, for example, a constitutively activeMEF2A/VP16, MEF2A/GAL4, MEF2B/VP16, MEF2B/GAL4, MEF2C/VP16, MEF2C/GAL4,MEF2D/VP16 or MEF2D/GAL4 fusion protein. A constitutively active MEF2polypeptide useful in the invention also can be, for example, a modifiedMEF2 polypeptide in which one or all of the p38 kinase phosphorylationsites in the MEF2 transactivation domain are substituted with anaspartic or glutamic acid residue. In a further embodiment, a method ofthe invention is practiced by administering to a subject a MEF2activator, whereby the p38 mitogen-activated protein kinase/myocyteenhancer factor 2 (MEF2) pathway is induced. Such a MEF2 activator canbe, for example, a nucleic acid molecule encoding p38α.

[0044] In another embodiment, a method of the invention for reducing theseverity of a neurologic condition by administering a caspase inhibitorin addition to administering the agent that induces the p38/MEF2pathway. It is understood that the caspase inhibitor can be administeredtogether with, prior to or following administration of the agent thatinduces the p38/MEF2 pathway. In one embodiment, a method of theinvention is practiced by administering a caspase inhibitor in additionto a constitutively active MEF2 polypeptide.

[0045] The invention further provides a method of protecting a neuronfrom cell death by inducing in the neuron the p38 mitogen-activatedprotein kinase/myocyte enhancer factor 2 (MEF2) pathway. Such a neuroncan be, for example, an adult neuron. A method of the invention can beuseful, for example, in protecting a neuron from apoptotic cell deathdue to an insult such as NMDA receptor-mediated toxicity, or oxidativeor nitrosative stress. In one embodiment, a method of the invention forprotecting a neuron from cell death is practiced by introducing into theneuron a nucleic acid molecule encoding a MEF2 polypeptide or an activefragment thereof. A method of the invention can be practiced, forexample, by introducing into a neuron a nucleic acid molecule encoding ahuman MEF2 polypeptide or an active fragment thereof and, in oneembodiment, is practiced by introducing into a neuron a MEF2Cpolypeptide or active fragment thereof, for example, a human MEF2Cpolypeptide or active fragment thereof.

[0046] In another embodiment, a method of the invention is practiced byintroducing into a neuron a nucleic acid molecule encoding aconstitutively active MEF2 polypeptide such as a constitutively activeform of MEF2A, MEF2B, MEF2C or MEF2D. In a further embodiment, theconstitutively active form of the MEF2 polypeptide is resistant tocaspase cleavage. A constitutively active MEF2 polypeptide can be, forexample, a chimera in which the native MEF2 activation domain isreplaced with a heterologous activation domain, for example, aconstitutively active MEF2A/VP16, MEF2A/GAL4, MEF2B/VP16, MEF2B/GAL4,MEF2C/VP16, MEF2C/GAL4, MEF2D/VP16 or MEF2D/GAL4 fusion proteins. Aconstitutively active MEF2 polypeptide also can be, for example, amodified MEF2 polypeptide in which one or all of the p38 kinasephosphorylation sites in the MEF2 transactivation domain are substitutedwith an aspartic or glutamic acid residue. In yet a further embodiment,a method of the invention is practiced by introducing into a neuron aMEF2 activator in order to induce the p38 mitogen-activated proteinkinase/myocyte enhancer factor 2 (MEF2) pathway in the neuron. ExemplaryMEF2 activators useful in the invention include p38α-encoding nucleicacid molecules.

[0047] In a further embodiment, a method of the invention for protectinga neuron from cell death is practiced by inducing the p38/MEF2 pathwayand further introducing a caspase inhibitor into the neuron. It isunderstood that the caspase inhibitor can be administered together with,prior to, or following induction of the p38/MEF2 pathway. In oneembodiment, a neuron is protected from cell death by introducing intothe neuron a constitutively active MEF2 polypeptide and a caspaseinhibitor.

[0048] The invention also provides a method of protecting a muscle cellfrom cell death by inducing in the muscle cell the p38 mitogen-activatedprotein kinase/myocyte enhancer factor 2 (MEF2) pathway. Such a musclecell can be, for example, an adult muscle cell. A method of theinvention can be useful, for example, in protecting a heart muscle cellfrom injury in a subject susceptible to heart attack (myocardialinfarction). In one embodiment, a method of the invention for protectinga muscle cell from cell death is practiced by introducing into themuscle cell a nucleic acid molecule encoding a MEF2 polypeptide or anactive fragment thereof. A method of the invention can be practiced, forexample, by introducing into the muscle cell a nucleic acid moleculeencoding a human MEF2 polypeptide or an active fragment thereof. In oneembodiment, a method of the invention is practiced by introducing intothe muscle cell a MEF2C polypeptide or an active fragment thereof, forexample, a human MEF2C polypeptide or an active fragment thereof.

[0049] In another embodiment, a method of the invention is practiced byintroducing into a muscle cell a nucleic acid molecule encoding aconstitutively active MEF2 polypeptide such as a constitutively activeform of MEF2A, MEF2B, MEF2C or MEF2D. In one embodiment, theconstitutively active form of the MEF2 polypeptide is resistant tocaspase cleavage. In yet a further embodiment, a method of the inventionfor protecting a muscle cell from cell death is practiced by introducinginto the muscle cell a constitutively active MEF2 polypeptide in whichthe native MEF2 activation domain is replaced with a heterologousactivation domain, for example, a constitutively active MEF2A/VP16,MEF2A/GAL4, MEF2B/VP16, MEF2B/GAL4, MEF2C/VP16, MEF2C/GAL4, MEF2D/VP16or MEF2D/GAL4 fusion proteins. A constitutively active MEF2 polypeptideuseful in protecting a muscle cell from cell death also can be, forexample, a modified MEF2 polypeptide in which one or all of the p38kinase phosphorylation sites in the MEF2 transactivation domain aresubstituted with an aspartic or glutamic acid residue. In yet anotherembodiment, a method of the invention is practiced by introducing into amuscle cell a MEF2 activator such as a p38α-encoding nucleic acidmolecule in order to induce the p38 mitogen-activated proteinkinase/myocyte enhancer factor 2 (MEF2) pathway in the neuron.

[0050] A method of the invention for protecting a muscle cell from celldeath also can be practiced by inducing the p38/MEF2 pathway and furtherintroducing a caspase inhibitor into the muscle cell. It is understoodthat the caspase inhibitor can be administered together with, prior to,or following induction of the p38/MEF2 pathway. In one embodiment, amuscle cell is protected from cell death by introducing into the musclecell a constitutively active MEF2 polypeptide and a caspase inhibitor.

[0051] The present invention further provides a method of generatingmuscle cells from progenitor cells by inducing in the progenitor cellsthe p38 mitogen-activated protein kinase/myocyte pathway andtransplanting the cells into a muscle cell environment, therebydifferentiating the progenitor cells to produce a population containingmuscle cells. In one embodiment, the progenitor cells are differentiatedto produce a cell population containing protected muscle cells. A methodof the invention for generating muscle cells can be useful, for example,for generating cardiac muscle following myocardial infarction,congestive heart failure, cardiomyopathy or other injury to hearttissue. Where cardiac muscle is to be generated, the cells can betransplanted into the heart wall, which provides the proper tissueenvironment for muscle cell differentiation. In one embodiment, a methodof the invention for protecting a muscle cell from cell death ispracticed by introducing into the muscle cell a nucleic acid moleculeencoding a MEF2 polypeptide or an active fragment thereof.

[0052] A method of the invention for generating muscle cells can bepracticed, for example, by introducing into a progenitor cell a nucleicacid molecule encoding a MEF2 polypeptide or an active fragment thereof.Such a MEF2 polypeptide can be, for example, a human MEF2 polypeptide.In one embodiment, a method of the invention for generating muscle cellsis practiced by introducing into a progenitor cell a MEF2C polypeptideor an active fragment thereof, for example, a human MEF2C polypeptide oran active fragment thereof.

[0053] In another embodiment, a method of the invention is practiced byintroducing into a progenitor cell a nucleic acid molecule encoding aconstitutively active MEF2 polypeptide, which can be, for example, aconstitutively active form of MEF2A, MEF2B, MEF2C or MEF2D. In a furtherembodiment, the constitutively active form of the MEF2 polypeptide isresistant to caspase cleavage. In yet a further embodiment, a method 0ofthe invention for generating muscle cells is practiced by introducinginto a progenitor cell a constitutively active MEF2 polypeptide in whichthe native MEF2 activation domain is replaced with a heterologousactivation domain. Exemplary constitutively active MEF2 polypeptidesinclude, for example, MEF2A/VP16, MEF2A/GAL4, MEF2B/VP16, MEF2B/GAL4,MEF2C/VP16, MEF2C/GAL4, MEF2D/VP16 and MEF2D/GAL4 fusion proteins andmodified MEF2 polypeptides in which one or all of the p38 kinasephosphorylation sites in the MEF2 transactivation domain are substitutedwith an aspartic or glutamic acid residue. In yet another embodiment, amethod of the invention for generating muscle cells is practiced byintroducing into a progenitor cell a MEF2 activator such as ap38α-encoding nucleic acid molecule in order to induce the p38mitogen-activated protein kinase/myocyte enhancer factor 2 (MEF2)pathway in the neuron.

[0054] In a further embodiment, muscle cells are generated by inducingthe p38/MEF2 pathway and further introducing a caspase inhibitor intothe progenitor cells. It is understood that the caspase inhibitor can beadministered together with, prior to, or following induction of thep38/MEF2 pathway. In one embodiment, a muscle cells are generatedaccording to a method of the invention by introducing into theprogenitor cells a constitutively active MEF2 polypeptide and a caspaseinhibitor.

[0055] Further provided by the invention is an isolated progenitor cellwhich contains an exogenous nucleic acid molecule encoding a MEF2polypeptide or an active fragment thereof. A progenitor cell of theinvention can contain, for example, a nucleic acid molecule encoding aMEF2 polypeptide, or active fragment thereof, operatively linked to aheterologous regulatory element. In one embodiment, the MEF2 polypeptideis a human MEF2 polypeptide or an active fragment thereof. In anotherembodiment, the MEF2 polypeptide is a MEF2C polypeptide or an activefragment thereof. In further embodiments, the MEF2 polypeptide is aconstitutively active MEF2 polypeptide, which can be, for example, aconstitutively MEF2A, MEF2B, MEF2C or MEF2D polypeptide and further canbe, for example, a constitutively active MEF2 polypeptide in which thenative MEF2 activation domain is replaced with a heterologous activationdomain. In yet a further embodiment, a constitutively active MEF2polypeptide is resistant to caspase cleavage. A constitutively activeMEF2 polypeptide useful in a progenitor cell of the invention can be,for example, a MEF2A/VP16, MEF2A/GAL4, MEF2B/VP16, MEF2B/GAL4,MEF2C/VP16, MEF2C/GAL4, MEF2D/VP16 or MEF2D/GAL4 fusion protein. Aconstitutively active MEF2 polypeptide also can be a modified MEF2polypeptide in which one or all of the p38 kinase phosphorylation sitesin the MEF2 transactivation domain are substituted with an aspartic orglutamic acid residue.

[0056] A progenitor cell useful in the invention can be a humanprogenitor cell such as a human stem cell and, if desired, can be aCD133-positive human progenitor cell. Progenitor cells useful in theinvention can be selected such that they are enriched for specificmarkers. Human progenitor cells useful in the invention include, forexample, CD133-positive human progenitor cells;CD133-positive/CD34-positive human progenitor cells;CD133-positive/CD34-negative human progenitor cells;CD133-positive/CD34-negative/CD45-negative;CD34-negative/CD38-negative/Lin-negative human progenitor cells; andCD34-positive/CD38-negative/ Lin-negative/Thy-1-negative humanprogenitor cells.

[0057] Progenitor cells useful in the invention include stem cells,which can be, for example, embryonic stem cells such as human embryonicstem cells. Progenitor cells useful in the invention also can be, forexample, human hematopoietic progenitor cells including the mostundifferentiated, pluripotent hematopoietic progenitor cells, which canbe denoted “hematopoietic stem cells.” In one embodiment, a progenitorcell of the invention is a human progenitor cell such as aCD133-positive/CD34-negative/Lin-negative human progenitor cellcontaining an exogenous nucleic acid molecule encoding a human MEF2Cpolypeptide or active fragment thereof. In still further embodiments, aprogenitor cell of the invention is a human progenitor cell such as aCD133-positive/CD34-negative/Lin-negative human progenitor cell thatcontains an exogenous nucleic acid molecule encoding a constitutivelyactive MEF2 polypeptide or an active fragment thereof.

[0058] As used herein, the term “stem cell” means a pluripotent celltype which can differentiate under the appropriate conditions to giverise to all cellular lineages. Thus, a stem cell differentiates toneuronal cells, hematopoietic cells, muscle cells, adipose cells, germcells and all other cellular lineages. A stem cell can be an embryonicstem cell. Where the term “hematopoietic stem cell” is used, it isunderstood that this term refers to cells that are committed to thehematopoietic lineage but which can differentiate to all cells of thehematopoietic lineage.

[0059] As used herein, the term “embryonic stem cell” is synonymous with“ES cell” and means a pluripotent cell type derived from an embryo whichcan differentiate to give rise to all cellular lineages. Examples ofcell markers that indicate a human embryonic stem cell include the Oct-4transcription factor, alkaline phosphatase, SSEA-4, TRA 1-60, and GCTM-2epitope as described in Reubinoff et al., supra, 2000. Examples of cellmarkers that indicate a differentiated neuronal cell includingneurofilament proteins, β-tubulin, Map2a+b, synaptophysin, glutamic aciddecarboxylase, TuJ1, SNAP 25, transcription factor Brn-3, and GABA_(A)α2 receptor subunit as described in Reubinoff et al., Nat. Biotech.18:399-404 (2000); Ghosh and Greenberg, Neuron 15:89-103 (1995); Bain etal., Devel. Biol. 168:342-357 (1995); and Williams et al., Neuron18:553-562 (1997).

[0060] The methods of the invention also can be used to differentiateprogenitor cells in the same manner as the disclosed methods fordifferentiating stem cells. As used herein, the term “progenitor cells”means any cells that are capable of differentiating into the desiredcell type such as a neuronal cell under the appropriate conditions.Progenitor cells can be multipotent or unipotent and can be stem cells,precursor cells, primary cells or established cells. Progenitor cellssuch as stem cells generally are distinct from neurons in that they lackneuronal markers such as the nuclear protein NeuN, neurofilament andmicrotubule-associated protein2 (MAP2) as well as the neuronal-likeprocesses characteristic of mature neurons. In one embodiment,progenitor cells are cells other than P19 embryonic carcinoma cells,which are cells from an established cell line that differentiates toneurons when treated with retinoic acid and to myocytes when treatedwith dimethylsulfoxide. In another embodiment, progenitor cells areprimary cells, which is a well known term in the art for cells which arederived directly from an organism and which have limited growth capacityin culture.

[0061] A progenitor cell useful in the invention can be multipotent orunipotent. As used herein in reference to a progenitor cell, the term“multipotent” is synonymous with “pluripotent” and means a progenitorcell capable of differentiating into two or more distinct lineages,including the neuronal lineage. Multipotent progenitor cells such asstem cells, which are generally nestin-positive cells, are distinguishedfrom unipotent precursor cells, which are generally Hu-positive cells.Expression of nestin and Hu can be determined, for example, byimmunocytochemistry as disclosed in Example II. A multipotent progenitorcell is capable of differentiating into at least three or more, four ormore, or five or more distinct lineages, including the neuronal lineage.

[0062] The methods of the invention are useful for differentiatingprogenitor cells to produce a population containing protected neuronalcells. As used herein, the term “neuronal cell” means a nerve cell andis characterized, in part, by containing one or more markers of neuronaldifferentiation. Such a marker can be, for example, neurofilament, NeuNor MAP2. A neuronal cell further generally is characterized ascontaining neuronal-like processes as shown in FIG. 6F.

[0063] The results disclosed herein indicate that the p38α/MEF2 cascadeprotects differentiating cells from death during neurogenesis. Thus, themethods of the invention rely, in part, on inducing the p38mitogen-activated protein kinase/myocyte enhancer factor 2 (p38 MAPkinase/MEF2) pathway in a progenitor cell. The p38 MAP kinase/MEF2pathway can be induced by any of a variety of means that result in anincrease in MEF2A, MEF2B, MEF2C or MEF2D expression or activity. Forexample, a MEF2 polypeptide can be phosphorylated and activated, therebyinducing the p38 MAP kinase/MEF2 pathway. p38α, for example, is a knownactivator, and, therefore, transfection of a p38α encoding nucleic acidmolecule or treatment with an agent that increases p38α expression oractivity can be used to induce the p38 MAP kinase/MEF2 pathway in amethod of the invention (see Example VIII). Thus, transcription factorsthat increase transcription of a MEF2 polypeptide or p38α; kinases orother proteins that activate a MEF2 polypeptide; or upstream effectorssuch as PAK-γ can be used to induce the p38 MAP kinase/MEF2 pathway. Onecan readily assay for induction of the p38 MAP kinase/MEF2 pathway byassaying for MEF2 binding activity and transcriptional activitydependent on the presence of the MEF2 binding site.

[0064] Induction of the p38 MAP kinase/MEF2 pathway also can be achievedusing a MEF2 activator, which is a small molecule that results inincreased expression or activity of a MEF2 polypeptide or which is amimetic or MEF2 function. A MEF2 activator can result in increasedexpression or activity of one or more MEF2 polypeptides, for example,may result in increased expression or activity of MEF2C withouteffecting expression or activity of MEF2A, MEF2B or MEF2D. Such a MEF2activator can be an organic chemical, drug, nucleic acid molecule,peptide, peptidomimetic, polypeptide or other naturally or non-naturallyoccurring organic molecule, and can be, for example, a MEF2 mimetic.Exemplary MEF2 activators are transcription factors that upregulate MEF2expression, molecules that compete for binding to a MEF2 inhibitor suchas Cabin1 or histone deacetylase, and kinases that activate MEF2polypeptides such as p38α. It is understood that a MEF2 activator can beuseful in any of the methods of the invention in which the p38mitogen-activated protein kinase/myocyte enhancer factor 2 (MEF2)pathway is induced. Thus, a MEF2 activator can be useful, for example,in differentiating progenitor cells to produce a cell populationcontaining protected neuronal cells, in reducing the severity of aneurologic condition, in protecting a neuron or muscle cell from celldeath, or in generating muscle cells.

[0065] MEF2 is normally sequestered in a transcriptionally inactivestate by Cabin1 (Youn et al., Science 286-790793 (1999)). Thus, a MEF2activator can be a factor that decreases expression of Cabin1 or afactor that promotes dissociation of MEF2 from Cabin1. Such a factor canbe, for example, a fragment of Cabin1 or a fragment of MEF2 thatcompetes for Cabin1 binding to MEF2, thereby dissociating MEF2 fromCabin1 and increasing the amount of active endogenous MEF2. Such MEF2activators can be identified by preparing and screening fragments ofCabin1 and MEF2 using routine methods.

[0066] MEF2 also is post-translationally regulated by class II histonedeacetylases, which bind the DNA-binding domain of MEF2 polypeptides (Luet al., PNAS 97:4070-4075 (2000)). MEF2 activity can be maximallystimulated only when repression by histone deacetylases is relieved, forexample, by calmodulin-dependent protein kinase signalling to theDNA-binding domain. Thus, a MEF2 activator can be a factor thatdecreases histone deacetylase expression or that promotes dissociationof MEF2 from histone deacetylase. Such a MEF2 activator can be, forexample, a fragment of histone deacetylase or a fragment of MEF2 thatcompetes for binding of MEF2 to histone deacetylase, therebydissociating histone deacetylase and increasing the amount of activeMEF2 polypeptide. Such a MEF2 activator can be identified by preparingand screening fragments of histone deacetylase and MEF2 using routinemethods.

[0067] An increase in either the p38α kinase or the big MAP kinase(Bmk1), also known as ERK5 kinase, increases MEF2 activity. Therefore, aMEF2 activator can be a molecule that increases the expression oractivity of p38α kinase, for example, a nucleic acid molecule encodingp38α (Matsumoto et al., J. Biol. Chem. 274:13954-13960 (1999)).Similarly, a MEF2 activator also can be a molecule that increases theexpression or activity of Bmk1/ERK5, for example, a nucleic acidmolecule encoding Bmk1/ERK5 (English et al., Journal of BiologicalChemistry 274:31588-31592 (1999); Kato et al., Nature 395:713-716(1998)).

[0068] In one embodiment, the p38 MAP kinase/MEF2 pathway is induced byintroducing a nucleic acid molecule encoding a MEF2 polypeptide into aprogenitor cell under conditions suitable for expression of the MEF2polypeptide in the cell. MEF2 polypeptides, which occur in a variety ofisoforms and alternatively spliced forms, are characterized, in part, asbelonging to the MADS-box family of transcriptional regulators. TheMADS-box is a 57 amino acid motif located at the extreme N-terminus ofMEF2 polypeptides (FIG. 1A). This motif serves as a minimal DNA-bindingdomain and, in conjunction with an adjacent 29-amino acid extensiondesignated the MEF2 domain, confers high-affinity DNA binding anddimerization (Molkentin et al., Mol. Cell. Biol. 16:2627-36 (1996).Within the MADS-box, MEF2 polypeptides share homology at severalinvariant residues with other members of the MADS-box family oftranscription factors, including serum response factor (SRF). Theseconserved residues are important for DNA sequence recognition. While theMEF2 domain is unique to MEF2 factors, other MADS-box proteins containdomains with analogous functions. In addition to its role in DNAbinding, the MADS-box mediates dimerization of MADS-box proteins, andthe MEF2 domain is important for interactions with accessory factors.MEF2 polypeptides can homo- and heterodimerize but cannot interact withother MADS-box factors, indicating that specific residues within theMADS-box that establish the dimerization interface are not conservedoutside the MEF2 family.

[0069] Vertebrate MEF2 polypeptides share about 50% amino acid identityoverall and about 95% similarity throughout the highly conservedMADS-box and MEF2 domain, whereas they are divergent in their C-terminalregions. MEF2 polypeptides from invertebrates also are highly homologousto vertebrate MEF2 polypeptides in the MADS-box and MEF2 domain. TheDrosophila MEF2 polypeptide, D-MEF2, binds the same DNA sequence as itsvertebrate counterparts and can activate transcription through the MEF2site in mammalian cells (Lilly et al., Proc. Natl. Acad. Sci. USA91:5662-66 (1994) and Nguyen et al., Proc. Natl. Acad. Sci USA91:7520-24 (1994)).

[0070] MEF2 polypeptides, like other MADS-box proteins, bind an A/T-richDNA sequence. The consensus MEF2 binding site is YTA(A/T)₄TAR. MEF2A,MEF2C and MEF2D have the same DNA binding specificity, whereas MEF2Bbinds the MEF2 consensus sequence with reduced affinity compared toother family members. Nucleotides flanking the MEF2 site have been shownto profoundly influence DNA binding (Yu et al., Genes Dev. 6:1783-98(1992); Andres et al., J. Biol. Chem. 270:23246-49 (1995); and FickettMol. Cell. Biol. 16:437-41 (1996)). Evidence suggests that the DNAbinding site is bent upon high affinity DNA binding (Meierhans et al.,Nucleic Acids Res. 25:4537-44 (1997)).

[0071] While the MADS-box and MEF2 domain are necessary and sufficientfor DNA binding, they lack transcriptional activity on their own. TheC-terminal regions of MEF2 polypeptides contain transcriptionalactivation domains and are subject to alternative splicing (FIG. 1A),with some exons present ubiquitously and others muscle- orneural-specific. In MEF2A, an acidic exon with the sequence SEEEELEL(SEQ ID NO:9) is specific to muscle and neural cells in which MEF2DNA-binding activity is detected and is absent in MEF2 transcripts froma variety of cell types in which MEF2A protein is not detected (Yu etal., Genes Dev. 6:1783-98 (1992)). MEF2 D contains a similar acidic exon(TEDHLDL; SEQ ID NO:10), which is present only in transcripts fromskeletal muscle, heart, and brain, and correlates with MEF2D-bindingactivity (Breitbart et al., Development 118:1095-106 (1993) and Martinet al., Mol. Cell. Biol. 14:1647-56 (1994)). The corresponding domain inMEF2C (SEDVDLLL; SEQ ID NO:11) is present in transcripts from skeletalmuscle only (McDermott et al., Mol. Cell. Biol. 13:2564-77 (1993)).Although the inclusion of these exons is not essential for DNA bindingactivity, their presence appears to correlate with high levels of MEF2DNA-binding activity. There is relatively little amino acid homologybetween the C-terminal regions of different MEF2 polypeptides, exceptfor the short acidic exons described above and fourserine/threonine-rich regions (FIG. 1B).

[0072] As used herein, the term “MEF2 polypeptide” means a polypeptidethat has MEF2 DNA binding activity in addition to activity as atranscriptional activator and includes polypeptides having substantiallythe amino acid sequence of MEF2A, MEF2B, MEF2C or MEF2D. Thus, a MEF2polypeptide can have, for example, substantially the amino acid sequenceof human MEF2A (SEQ ID NO:2) shown in FIG. 2, human MEF2B (SEQ ID NO:4)shown in FIG. 3; human MEF2C (SEQ ID NO:6) shown in FIG. 4, or humanMEF2D (SEQ ID NO:8) shown in FIG. 5. A MEF2 polypeptide includes a MADSdomain, a MEF2 domain and a transcriptional activation domain. It isunderstood that, while the MADS domain and MEF2 domains of a MEF2polypeptide will be similar in structure to the MADS domain and MEF2domain of a naturally occurring MEF2 polypeptide such as human MEF2C(SEQ ID NO:6), the transcriptional activation domain of a MEF2polypeptide may be structurally unrelated and can be, for example, asynthetic transcriptional activation or a heterologous transcriptionalactivation domain derived, for example, from VP16 or GAL4. One skilledin the art appreciates that a fragment of a MEF2 polypeptide thatretains MEF2 DNA binding activity and transcriptional activity also canbe useful in the methods and compositions of the invention.

[0073] The term MEF2 polypeptide encompasses a polypeptide having thesequence of a naturally occurring human MEF2A polypeptide (SEQ ID NO:2),naturally occurring human MEF2B polypeptide (SEQ ID NO:4), naturallyoccurring human MEF2C polypeptide (SEQ ID NO:6) or naturally occurringhuman MEF2D polypeptide (SEQ ID NO:8) and is intended to include relatedpolypeptides having substantial amino acid sequence similarity to SEQ IDNOS:2, 4, 6 or 8. Such related polypeptides typically exhibit greatersequence similarity to hMEF2A, hMEF2B, hMEF2C or hMEF2D than to otherMADS box proteins such as serum response factor (SRF) and includespecies homologs such as primate, mouse, rat and D. rerio homologs,alternatively spliced forms, and isotype variants of the proteins shownin FIGS. 2 through 5.

[0074] As used herein, the term MEF2 polypeptide describes polypeptidesgenerally including an amino acid region with greater than about 60%amino acid sequence identity in the combined MADS and MEF2 domains withhMEF2A (SEQ ID NO:2), hMEF2B (SEQ ID NO:4), hMEF2C (SEQ ID NO:6) orhMEF2D (SEQ ID NO:8). In particular, a MEF2polypeptide can have greaterthan about 65% amino acid identity, preferably greater than about 70%amino acid identity, more preferably greater than about 75% amino acididentity, still more preferably greater than about 80% amino acididentity and most preferably greater than about 85%, 90% or 95% aminoacid identity with the combined MADS and MEF2 domains of SEQ ID NOS:2,4, 6 or 8.

[0075] As used herein, the term “substantially the amino acid sequence,”when used in reference to a MEF2 polypeptide or fragment thereof, isintended to mean a polypeptide or fragment having an identical aminoacid sequence, or a polypeptide, fragment or segment having a similar,non-identical sequence that is considered by those skilled in the art tobe a functionally equivalent amino acid sequence. For example,polypeptide including substantially the same amino acid sequence ashuman MEF2C (SEQ ID NO:6) can have an amino acid sequence identical tothe sequence of human MEF2C (SEQ ID NO:6) shown in FIG. 4, or a similar,non-identical sequence that is functionally equivalent. An amino acidsequence that is “substantially the amino acid sequence” can have one ormore modifications such as amino acid additions or substitutionsrelative to the amino acid sequence shown, provided that the modifiedpolypeptide retains the ability to bind the MEF2 binding site and toactivate transcription.

[0076] Therefore, it is understood that limited modifications can bemade without destroying the biological function of a MEF2 polypeptide orfragment useful in the invention. For example, minor modifications ofhMEF2C (SEQ ID NO:6) that do not destroy polypeptide activity also fallwithin the definition of a MEF2 polypeptide. Similarly, minormodifications of human MEF2A, -B, -C or -D that do not destroypolypeptide activity fall within the definition of a MEF2 polypeptide.Also, for example, genetically engineered fusion proteins that retainthe DNA binding and transcriptional activation activity of a MEF2polypeptide fall within the meaning of the term “MEF2 polypeptide” asused herein.

[0077] It is understood that minor modifications of primary amino acidsequence can result in polypeptides which have substantially equivalentor enhanced function as compared to the MEF2 polypeptides shown in FIGS.2 through 5. These modifications can be deliberate, as throughsite-directed mutagenesis, or can be accidental such as through mutationin hosts harboring an encoding nucleic acid. All such modifiedpolypeptides are included in the definition of a MEF2 polypeptide aslong as MEF2 DNA binding activity and transcriptional activationactivity are retained. Further, various molecules can be attached to aMEF2 polypeptide, for example, other polypeptides, carbohydrates,lipids, or chemical moieties. Such modifications are included within thedefinition of each of the polypeptides of the invention.

[0078] While native MEF2 polypeptides are activated throughphosphorylation, for example, by p38 MAP kinase, constitutively activeforms of MEF2 do not require such phosphorylation for activation. Any ofthe methods of the invention can be practiced using a constitutivelyactive MEF2 polypeptide to induce the p38/MEF2 pathway.

[0079] As used herein in reference to a MEF2 polypeptide, the term“constitutively active” means a MEF2 polypeptide that hastransactivation activity which is less dependent upon phosphorylationthan the corresponding wild type MEF2 polypeptide. A constitutivelyactive MEF2 polypeptide can have transactivation activity that isindependent of phosphorylation. As disclosed herein, a MEF2 polypeptidecan be cleaved by a caspase to produce a dominant negative form of aMEF2 polypeptide having pro-apoptotic activity. In one embodiment, aconstitutively active form of a MEF2 polypeptide is resistant to caspasecleavage.

[0080] A constitutively active MEF2 polypeptide can include, forexample, a heterologous transactivation domain in addition to, or inplace of, the native MEF2 transactivation domain. A constitutivelyactive MEF2 polypeptide can be, for example, a MEF2A, MEF2B, MEF2C orMEF2 D polypeptide containing a GAL4 or VP16 transactivation domain inaddition to, or in place of, the native MEF2 transactivation domain. Inspecific embodiments, a constitutively active MEF2 polypeptide is achimera in which the native MEF2 activation domain is replaced with aheterologous activation domain, for example, a constitutively activeMEF2A/VP16, MEF2A/GAL4, MEF2B/VP16, MEF2B/GAL4, MEF2C/VP16, MEF2C/GAL4,MEF2D/VP16 or MEF2D/GAL4 fusion protein.

[0081] A constitutively active MEF2 polypeptide also can be a MEF2polypeptide in which the native activation domain is modified such thattransactivation does not depend on phosphorylation. A constitutivelyactive MEF2 polypeptide can have, for example, one or modifiedphosphorylation sites within the transactivation domain, for example,one or more serine/threonine to aspartic acid/glutamic acid amino acidsubstitutions within the transactivation domain. See, for example,Watson et al., J. Neurosci. 18:751-762 (1998), which demonstrates thatmutation of the Jun kinase phosphorylation site in c-Jun to asparticacid produces a constitutively active c-Jun polypeptide that isindependent of Jun kinase.

[0082] MEF2A is phosphorylated at Thr312, Thr319and Ser453 within thetransactivation domain, and MEF2C is phosphorylated at Thr293, Thr300and Ser387 within the transactivation domain (Han et al., Nature386:296-299 (1997); and Zhao et al., Mol. Cell. Biol. 19:21-30 (1999)).Thus, a constitutively active human MEF2A polypeptide can contain, forexample, one or more amino acid substitutions such that one or all ofThr312, Thr319and Ser453 are replaced with aspartic or glutamic acid. Inaddition, a constitutively active human MEF2C polypeptide can contain,for example, one or more amino acid substitutions such that one or allof Thr293, Thr300 and Ser387 are replaced with aspartic or glutamicacid. It is understood that analogous phosphorylation sites in MEF2B andMEF2D and other species homologs of MEF2A and MEF2C can be similarlymodified to produce a constitutively active MEF2 polypeptide.

[0083] A variety of routine assays can be used to confirm constitutiveactivity of a MEF2 polypeptide including cotransfection assays usingcerebrocorticol neurons, where constitutive activity is indicated byreporter activity significantly greater than wild type MEF2transcriptional activity. For example, cerebrocorticol neurons can becultured for about five hours using Lipofectamine2000 with the MEF2expression vector and a luciferase reporter vector such as pGL2MEF2LUC,and luciferase activity determined by standard means.

[0084] As stated above, a MEF2 polypeptide can be cleaved by a caspaseto produce a dominant negative form of a MEF2 polypeptide havingpro-apoptotic activity. Thus, in the presence of an activated caspase,protective MEF2 activity can be enhanced by a caspase inhibitor. Thus,in one embodiment of the invention, induction of the the p38mitogen-activated protein kinase/myocyte enhancer factor 2 (MEF2)pathway is combined with treatment of the cell by a caspase inhibitor.

[0085] A variety of caspase inhibitors are useful in the inventionincluding, for example, nucleic acids, polypeptides, peptides,peptidomimetics and non-peptide inhibitors such as small molecule drugsknown in the art. As used herein, the term “caspase inhibitor” means anymolecule that binds to and inhibits the activity of one or morecaspases. Caspase inhibitors useful in the methods of the inventiongenerally are cell permeable and have inhibitory activity in vivo andinclude viral and cellular gene products as well as synthetic inhibitorssuch as synthetic small molecules (Ekert et al., Cell Death andDifferentiation 6:1081-1086 (1999)).

[0086] Such a caspase inhibitor can be a general, or non-selective,caspase inhibitor as well as a selective inhibitor. Selective inhibitorsdo not inhibit non-caspase cysteine proteases or serine proteases.Non-selective caspase inhibitors, which also inhibit one or morenon-caspase protease inhibitors, include, for example, the cysteineprotease inhibitor iodoacetamide. A caspase inhibitor also can beselective for one or more specific caspases. A caspase inhibitor canselectively inhibit caspase-3 or caspase-7 or a combination thereof andcan be combined, for example, with a nucleic acid molecule encoding aMEF2C polypeptide, or an active fragment thereof. An exemplary caspaseinhibitor which is selective for caspases-3 and -7 is a non-peptideinhibitor such as a isatin sulfonamide (see, for example, Lee et al., J.Biol. Chem. 275:16007-16014 (2000)). A selective caspase inhibitor alsocan be selective for caspase-3, caspase-6, caspase-7 or caspase-8, orany combination thereof, and can be combined, for example, with anucleic acid molecule encoding a MEF2A polypeptide, or an activefragment thereof.

[0087] A caspase inhibitor can be, for example, the cytokine responsemodifier A (CrmA) polypeptide, or an encoding nucleic acid molecule,which inhibits caspases-1 and -8; or the p35 baculovirus protein, or anencoding nucleic acid molecule, which inhibits caspases-1, -3, -6, -7,-8 and -10 but does not inhibit non-caspase cysteine proteases or serineproteases (Clem et al., Science 254:1388-1390 (1991)). A caspaseinhibitor also can be an inhibitor of apoptosis protein (IAP) or anencoding nucleic acid molecule. IAPs useful as caspase inhibitors in amethod of the invention include XIAP and Survivin, which inhibitcaspases-3 and -7.

[0088] A caspase inhibitor also can be a synthetic caspase inhibitorsuch as a pseudosubstrate which acts as reversible or irreversiblecompetitive inhibitors of caspases. Active site mimetic peptide ketonesare useful, for example, as selective caspase inhibitors. Such caspaseinhibitors include, for example, benzylcarbonyl(z)-VAD-fluoromethylketone (fmk), z-VAD-fmk/chloromethylketone (CMK),z-DEVD-fmk/cmk; and z-D-cmk. Additional caspase inhibitors include thehalomethyl ketone-linked peptide YVAD, Ac-WEHD-CHO, Ac-DEVD-CHO,Ac-YVAD-CHO, t-butoxycarbonyl-IETD-CHO, and t-butoxycarbonyl-AEVD-CHO(Ekert et al., supra, 1999). The skilled person understands that theseand other caspase inhibitors can be useful in the invention. See, forexample, Nicholson, Nature 407:810-816 (2000), WO 00/55114, andGarcia-Calvo et al., J. Biol. Chem. 273:32608-32613 (1998)).

[0089] As used herein in reference to a neuronal cell, the term“protected” means a cell that is induced to undergo neurogenesis and ismore resistant to apoptotic cell death than a cell in which the p38 MAPkinase/MEF2 pathway is not induced, or is induced to a lesser extent.

[0090] Thus, a population containing protected neuronal cells willexhibit less apoptosis than a population that does not contain“protected” neuronal cells.

[0091] The percentage of apoptotic cells in a population can bedetermined by a variety of assays well known in the art. Such methodsinclude light microscopy for determining the presence of one or moremorphological characteristics of apoptosis, such as condensed or roundedmorphology, shrinking and blebbing of the cytoplasm, preservation ofstructure of cellular organelles including mitochondria, andcondensation and margination of chromatin. The percentage of apoptoticcells also can be determined by assaying apoptotic activity usingterminal deoxytransferase-mediated (TdT) dUTP biotin nick end-labeling(TUNEL) (Gavriel et al., J. Cell Biol. 119:493 (1992); Gorczyca et al.,Int. J. Oncol. 1:639 (1992); Studzinski (Ed.), Cell Growth andApoptosis, Oxford: Oxford University Press (1995)). ApopTag™ (ONCOR,Inc., Gaithersburg, Md.) is a commercially available kit foridentification of apoptotic cells using digoxygenin labeling. Inaddition, apoptotic cells can be identified by detecting characteristicnucleosomal DNA fragments using agarose gel electrophoresis (Studzinski,supra, 1995; Gong et al., Anal. Biochem. 218:314 (1994)) or using DNAfilter elution methodology to detect apoptosis-associated DNAfragmentation (Bertrand et al., Drug Devel. 34:138 (1995)). One skilledin the art understands that these, or other assays for apoptosis, can beperformed using methodology routine in the art.

[0092] In the methods of the invention, progenitor cells are contactedwith a differentiating agent. In one embodiment, the differentiatingagent is retinoic acid, for example, all trans-retinoic acid. In anotherembodiment, the differentiating agent is neurotrophic factor 3,epidermal growth factor, insulin-like growth factor 1 or aplatelet-derived growth factor.

[0093] As used herein, the term “differentiating agent” means anaturally occurring or synthetic cytokine, growth factor or othercompound that causes or enhances a progenitor cell to have one or morecharacteristics of a neuronal cell. A differentiating agent useful inthe invention can be, for example, retinoic acid such as all-transretinoic acid; neurotrophic factor 3 (NT3); epidermal growth factor(EGF); insulin-like growth factor-1 (IGF-1); platelet derived growthfactor (PDGF), or a combination of two or more of these factors. Forexample, EGF, IGF-1 and PDGF can be used together as a differentiatingagent. Basic fibroblast growth factor (bFGF) or another factor thatenhances proliferation of precursor cells can optionally be used priorto treating with a differentiating agent such as EGF, IGF-1 and PDGF.One skilled in the art understands that one or more factors such asbrain-derived neurotrophic factor (BDNF) also can be added to promoteneuronal cell survival.

[0094] For use in the methods or compositions of the invention,embryonic stem cells can be obtained from a variety of mammalsincluding, for example, mice, cows, primates and humans by methods wellknown in the art. For example, murine embryonic cells can be isolatedfrom a mouse as described in Forrester et al., Proc. Natl. Acad. Sci.USA 88:7514-7517 (1991) or Bain et al., Devel. Biol. 168:342-357 (1995).Briefly, two-stage cell embryos can be isolated from fertilized femalemice about 45 hours after injection with human chorionic gonadotropin.The two blastomeres can be fused by electrical impulse and cultured inM16 medium until the four cell stage is reached. ES cells can be grownon gelatin coated tissue culture flasks in DMEM (Dulbeco's modifiedEagle's medium) containing high glucose and 1-glutamine (BRL)supplemented with 10% fetal bovine serum, 10% newborn calf serum,nucleosides stock, 1000 units/ml leukemia inhibitory factor, and 0.1 mM2-mercaptoethanol.

[0095] Embryonic stem cells can be isolated from primates as describedin Thomson (U.S. Pat. No. 5,843,780). Briefly, blastocysts can beremoved from fertilized female monkeys 6-8 days after onset ofovulation, treated with pronase (Sigma) to remove the zona pellucida,rabbit anti-rhesus monkey spleen cell antiserum (for blastocysts fromrhesus monkeys) and guinea pig complement (Gibco BRL), and washed inDMEM. The inner cell mass (ICM) can be removed from the lysed blastocystwith a pipette and plated on mouse gamma radiation inactivated embryonicfibroblasts. After 7 to 21 days the ICM derived masses can be removedwith a micropipette, treated with 0.05% trypsin-EDTA (Gibco BRL) and 1%chicken serum, and replated on embryonic feeder cells. Coloniesdemonstrating ES morphology, characterized by compact colonies with ahigh nucleus to cytoplasm ratio and prominent nucleoli, can then besplit as described above. The ES cells can be split by trypsinization orexposure to Dulbeco's phosphate buffered saline containing 2 nM EDTAevery 1-2 weeks when cultures become dense.

[0096] Embryonic stem-like cells also can be isolated from cows asdescribed in Cibelli et al., Nat. Biotech. 16:642-646 (1998). Briefly,oocytes can be removed from freshly slaughtered cows and placed inmaturation medium M199 (Gibco), 10% fetal calf serum (FCS), 5 ug/mlbovine leutinizing hormone (Nobl) and 10 ug/ml pen-strep (Sigma) for 22hours at 38.5SC. Oocytes can then be fertilized in vitro and cultured onmouse embryonic fibroblast feeder layers and CR2 with 6 mg/ml BSA untilthey reach the blastocyst stage. ES cells can be isolated from theblastocyst by mechanical removal of the zona pellucida and trophoblastwith a 22 gauge needle and placed under mouse embryonic fibroblastfeeder layers for one week. A small colony of the resulting cell masscan be removed and cultured on top of gamma irradiation inactivatedmouse embryonic fibroblast feeder layer as cultures become dense.

[0097] Embryonic stem cells can be isolated from human blastocysts asdescribed in Reubinoff et al., Nat. Biotech. 18:399-404 (2000). Briefly,fertilized oocytes can be cultured to the blastocyst stage and the zonapellucida digested by pronase (Sigma). The inner cell mass can beremoved by immunosurgery with anti-human serum antibody (Sigma) andexposure to Guinea pig complement (BRL), and cultured on a mitomycin Cmitotically inactivated mouse embryonic feeder cell layer in DMEM (BRL)supplemented with 20% fetal bovine serum (FBS, Hyclone) 0.1 mM2-mercaptoethanol, 1% non essential amino acids, 2 nM glutamine, 50units/ml penicillin and 50 ug/ml streptomycin (BRL)and 2,000 units/mlrecombinant leukemia inhibitory factor. Cell mass clumps can be removedwith a micropipette and replated on fresh feeder layer every six toeight days.

[0098] Human stem cells can be obtained, for example, from cord blood,which is highly enriched in primitive cells and contains aCD133-positive/ CD34-positive population. These cells can be efficientlyisolated by methods well known in the art, for example, the MiltinylMACS system. If desired, the CD133-positive/CD34-positive population canbe expanded by culturing in vitro with Flt3L+TPO to produce as much asan 160-fold expansion in long-term culture potential and a 2×10⁶ foldexpansion in the number of progenitor cells.

[0099] Human progenitor cells useful in the invention include humanembryonic stem cells, human hematopoietic stem cells and otherprogenitor cells isolated from adult human blood or from cord blood ofnewborn infants. In one embodiment of the invention, the progenitor cellpopulation is enriched in CD133 (AC133)-positive/CD34-positiveprogenitor cells. In a further embodiment of the invention, theprogenitor cell population is enriched in CD133-positive/CD34-negativeprogenitor cells. Such specific progenitor cell populations can beisolated, for example, with magnetic-activated cell sorting,fluorescence-activated cell sorting (FACS), or related methods wellknown in the art as described further below. It further is understoodthat in vitro expansion of progenitor or stem cells such as humanprogenitor or stem cells can be performed, if desired, with one or moreof the following factors: SCF, IL-3, IL-6, flt3L, LIF, IL-11, TGF-β,TPO, EGF and bFGF, which are commercially available, for example, fromBiosource (Camarillo, Calif.), R & D Systems (Minneapolis, Minn.) andChemicon (Temecula, Calif.). Various protocols for expansion and usefulconcentrations of particular factors are well known in the art.

[0100] In one embodiment, human progenitor cells are obtained fromperipheral blood. Donors can be treated with recombinant human G-CSF(rhG-CSF), such as Neupogen (Amgen; Thousand Oaks, Calif.), orrecombinant human GM-CSF (rhGM-CSF), such as Leukine (Immunex; Seattle,Wash.), or both. In a further embodiment, the human progenitor cells areprimitive cells characterized as CD34+, Thy-/dim, CD38−, which can beobtained, if desired, from G-CSF or GM-CSF treated to donors to increaselong-term culture potential. In one embodiment, human progenitor cellsare CD34+, Thy-/dim, CD38− cells obtained from donors treated with G-CSFin combination with GM-CSF.

[0101] Methods well known in the art can be used to collect progenitorcells from human peripheral blood or cord blood. Apheresis can be usedto collect white blood cells, for example, four to five days followingtreatment with G-CSF, GM-CSF or a combination of G-CSF and GM-CSF,generally yielding 4×10⁶ CD34-positive cells/kg of body weight.

[0102] A Ceprate SC immunoaffinity column commercially available fromCellpro (Bothell, Wash.) can be used to isolate a CD133-positiveprogenitor cell population for use in a method of the invention. Thedesired cell population binds the column matrix via a biotin conjugatedantibody linked to the column matrix and is released by mechanicalshaking. Ceprate SC immunoaffinity can be used to yield about 50%CD34-positive cells with about 16-99% purity.

[0103] CD133-positive human progenitor cells also can be isolated, forexample, using an Isolex 300 magnetic cell separator (Baxter HealthcareCorporation; Deerfield, Ill.), which relies on mouse monoclonal IgGlantibodies and magnetic beads coated with anti-mouse IgGl antibody.Release of the progenitor cells by peptidase treatment yields about 50%CD34-positive cells with 33-100% purity.

[0104] Additional art-accepted procedures for isolation of human stemand progenitor cells include the magnetic activated cell sorting system(MACS) commercially available from Miltenyi Biotech (Auburn, Calif.). Inthis sorting system, small magnetic beads coated with secondary antibodyare bound to the primary antibody-treated cells and retained on aferromagnetic matrix column by a strong magnet. Cells are released byremoval of the magnet to give greater than 50% recovery and greater than90% purity of the desired cells.

[0105] Fluorescence-activated cell sorting (FACS) also is a well knownmethod that can be used to isolate the desired progenitor or stem cellpopulation. Using this methodology, cells are selected by attachment offluorescent-conjugated antibodies to give greater than 90% purity of therecovered stem or progenitor cells.

[0106] If desired, isolated stem or progenitor cells can be assayed forthe ability to repopulation bone marrow of a sublethally irradiatednonobese diabetic/severe combined immunodeficient (NOD-SCID) mouse,using methods well known in the art, as described, for example, inMiyoshi et al., Science 283:682-686 (1999).

[0107] In one embodiment, progenitor cells useful in the invention arehuman CD34-negative bone marrow cells such asCD133-positive/CD34-negative cells. In a further embodiment, theprogenitor cells are CD34-negative/Lin-negative cells. Such cells canhave characteristics of stromal cells and are capable, for example, ofrepopulating the bone marrow of NOD/SCID mice following sublethalirradiation. In one embodiment, progenitor cells useful in the inventionare CD133-positive/CD34-negative/Lin-negative cells.

[0108] Methods of preparing progenitor or stem cell populations enrichedfor particular markers are well known in the art. For example, aCD133-positive/CD34-positive hematopoietic stem and progenitor cells canbe prepared as set forth in Yin et al., Blood 90:5002-5012 (1997);CD133-positive/CD34 negative/CD45-negative progenitor cells can beprepared as described, for example, in Uchida et al., Proc. Natl. Acad.Sci., USA 97:14720-14725 (2000). In addition,CD34-negative/CD38-negative/Lin-negative human hematopoietic stem cellsand CD34-positive/CD38-negative/Lin-negative/Thy-1-negativehematopoietic stem cells can be prepared, for example, as described inBhatia et al., Nature Medicine 4:1038-1045 (1998).

[0109] In the methods of the invention, progenitor or stem cells such asembryonic stem cells are contacted with a differentiating agent toinduce differentiation of the cells along the neuronal pathway. Methodsfor differentiating embryonic stem cells by growth of the cells to highdensity are described in Reubinoff et al., Nat. Biotech. 18:399-404(2000). Methods differentiating expanded CNS cells by initial growth inthe presence of a mitogen such as basic fibroblast growth factor (bFGF)followed by removal of bFGF are described in Johe et al. Genes Develop.10:3129-3140 (1996). Induction of neurogenesis by addition of growthfactors can be achieved with platelet derived growth factor (PDGF)including for example PDGF-AA, PDGF-AB or PDGF-BB administered in theabsence of bFGF as described in Johe et al., supra, 1996. Induction ofneuronal differentiation can also be achieved in vitro by removal offibroblast growth factor-2 and subsequent addition of insulin likegrowth factor-1, heparin or neurotrophin-3as described in Brooker etal., J. Nerosci. Res. 59:332-341 (2000) and Ghosh and Greenberg, Neuron15:89-103 (1995); addition of platelet-derived growth factor asdescribed in Williams et al., Neuron 18:553-562 (1997); addition ofinsulin like growth factor-1 alone or in combination with brain derivedneurotrophic factor as described in Arsenijevic and Weiss, J Neurosci.18:2118-2128 (1998); and exposure to retinoic acid as described in Bainet al. Devel. Biol. 168:342-357 (1995).

[0110] In a preferred embodiment, a nucleic acid molecule encoding aMEF2 polypeptide is introduced into a progenitor cell such as anembryonic stem cell. A variety of methods are known in the art forintroducing a nucleic acid molecule into a progenitor cell such as anembryonic stem cell. Such methods include microinjection,electroporation, lipofection, calcium-phosphate mediated transfection,DEAE-Dextran-mediated transfection, polybrene- or polylysine-mediatedtransfection, and conjugation to an antibody, gramacidin S. artificialviral envelopes or other intracellular carriers such as TAT. Forexample, embryonic stem cells can be transformed by microinjection asdescribed in Cibelli et al., Nat. Biotech. 16:642-646 (1998) or Lamb andGearhart, Cur. Opin. Gen. Dev. 5:342-348 (1995); by lipofection asdescribed in Choi (U.S. Pat No. 6,069,010) or Lamb and Gearhart, Cur.Opin. Gen. Dev. 5:342-348 (1995); by electroporation as described inCurrent Protocols in Molecular Biology, John Wiley and Sons, pp9.16.4-9.16.11 (2000) or Cibelli et al., Nat. Biotech. 16:642-646(1998); or by fusion with yeast spheroplasts Lamb and Gearhart, Cur.Opin. Gen. Dev. 5:342-348 (1995). A MEF2 polypeptide also can bedelivered to stem or progenitor cells as a TAT/MEF2 polypeptide fusionby techniques well known in the art as described in Nagahara et al.,Nature Medicine 4:1449-1452 (1998).

[0111] Viral vectors can be particularly useful for introducing anucleic acid molecule encoding a MEF2 polypeptide in a method of theinvention; such vectors include, for example, retroviral vectors,lentiviral vactors, adenoviral vectors and adeno-associated vectors(AAV), herpesvirus vectors (see, for example, Kaplitt and Loewy, ViralVectors: Gene Therapy and Neuroscience Applications Academic Press, SanDiego, Calif. (1995); Chang, Somatic Gene Therapy CRC Press, Boca Raton,Fla. (1995)). Lentiviral, retroviral and adeno-associated vectors can beuseful, for example, for permanent expression, and adenovirus andherpesvirus can be used to achieve transient expression lasting forseveral months to about one year. It is understood that both permanentand transient expression can be useful in a method of the invention andin producing a stem or progenitor cell of the invention.

[0112] It is understood by those skilled in the art of gene therapy thata progenitor cell also can be engineered to express one or more geneproducts that are therapeutically useful. For example, for treatment ofParkinson's disease, a progenitor cell such as an embryonic stem cellcan express, for example, the catecholamine enzyme tyrosine hydroxylase,thereby increasing dopamine-β-hydroxylase activity upon intracerebralgrafting (Jiao et al., Nature 362:450 (1993); see, also, Dhawan et al.,Science 254:1509 (1991); and Barr and Leiden, Science 254:1507 (1991)).Similarly, for treatment of Alzheimer's disease, a progenitor cell canexpress a nucleic acid molecule encoding nerve growth factor, therebypromoting cell survival of the cholinergic neurons that are typicallylost in Alzheimer's disease (Rosenberg et al., Science 242:1575-1578(1988)). In a similar manner, a progenitor cell can express encephalinfor treatment of neuropathic disorders involving intractable pain. Oneskilled in the art recognizes that these and other combinations areencompassed by the methods of the invention for differentiatingprogenitor cells to produce a population containing protected neuronalcells.

[0113] A progenitor cell such as an ES cell further can be engineered toexpress one or more anti-apoptotic gene products such as a member of theBcl-2 family, for example, Bcl-2 (Anderson, Trends Pharm. Sci. 18:51(1997) or Bcl-X_(L); and Gross and et al., Genes Dev. 13:1899-1911(1999)), or a member of the inhibitor of apoptosis (IAP) family such asc-IAP-1, c-IAP-2, XIAP or NIAP (Deveraux and Reed, Genes Dev. 13:239-252(1999)). A progenitor cell further can be optionally engineered toexpress a basic helix-loop-helix protein (bHLH), especially a bHLHprotein naturally expressed in neuronal cells such as Mash-1, which canfunctionally interact with a MEF2 polypeptide (Mao and Nadal-Ginard, J.Biol. Chem. 271:14371-14375 (1996); Black et al., J. Biol. Chem.271:26659-26663 (1996). A progenitor cell such as an ES cell also can beengineered to express one or more factors that promote differentiationincluding, for example, neuroD, neuroD2, neuroD3, neurogenin1,neurogenin2, neurogenin3, MATH1 or MATH2 (Lee, Curr. Onin. Neurobiol.7:13-20 (1997)). Such a factor can be expressed instead of or inaddition to application of the differentiating agent to the progenitorcell.

[0114] Previous methods of producing neuronal cells have suffered fromthe shortcoming that the populations produced are heterogenous andcontain relatively few neurons. A method of the invention isadvantageous in that it can be used to produce a population containingprotected neuronal cells containing a large proportion of neuronalcells, for example, at least 50% neuronal cells. In other embodiments,the population produced includes at least 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or more neuronal cells. The proportion of neuronal cellscan be determined by assaying for one or more characteristic neuronalmarkers such as the presence of NeuN, neurofilament or MAP2.

[0115] A method of the invention for differentiating progenitor cells toproduce a population containing protected neuronal cells can optionallyinclude the step of transplanting into a patient cells treated to inducethe p38/MEF2 pathway. In a method of the invention, cells can betransplanted, for example, into the brain, eye (retina) or spinal cordafter neuronal injury or damage. Thus, cells treated to induce thep38/MEF2 pathway can be transplanted into a patient having or at riskof, for example, stroke or a neurodegenerative disease such asAlzheimer's disease; Huntington's disease; amyotrophic lateralsclerosis; Parkinson's disease; epilepsy; brain or spinal cord trauma;multiple sclerosis; optic neuropathy such as glaucoma; infection of thecentral nervous system; multiple system atrophy affecting the brain; oranother acute or chronic neurodegenerative condition. Upontransplantation, the cells begin to differentiate or continuedifferentiating to produce a cell population containing protectedneuronal cells.

[0116] As used herein, the term “patient” means any animal containingneurons, for example, a mammal such as a mouse, rat, dog, primate orhuman. A patient typically suffers from or is at high risk of developinga neurodegenerative disorder such as Parkinson's disease, Huntington'sdisease, Alzheimer's disease, amyotrophic lateral sclerosis or multiplesclerosis; hypoxia-ischemia (stroke); epilepsy; head or spinal cordinjury; optic neuropathies including glaucoma and macular degeneration,and disorders of photoreceptor degeneration such as retinitispigmentosa; metabolic, mitochondrial or infectious brain abnormalitiessuch as encephalitis, or suffers from neuropathic pain (see, forexample, Lipton and Rosenberg, New Engl. J. Med. 330: 613 (1994)).

[0117] Cells can be transplanted into a patient, for example, into thebrain or spinal cord using well known methods for transplanting or“grafting” neurons as described, for example, in McDonald et al., Nat.Med. 5:1410-1412 (1999), and summarized in Dunnett et al., Brit. Med.Bulletin 53:757-776 (1997). Methods for preventing or amelioratingrejection, for example, using cyclosporinA treatment, also are known inthe art.

[0118] Those skilled in the art understand that the steps of contactingthe progenitor cells with a differentiating agent and inducing the p38MAP kinase/MEF pathway can be performed in any order or simultaneously.It further is understood that a progenitor population in which the p38MAP kinase/MEF2 pathway has been induced can be transplanted into apatient prior to, during or after differentiation of the progenitorcells into neuronal cells. In one embodiment, cells are transplantedprior to or during differentiation. Where cells are transplanted priorto differentiation, the neuronal environment can drive the cells intothe desired neuronal cell type, rather than, for example, muscle cellsdue to the presence of the appropriate environmental cues. In view ofthe above, it is clear that differentiation can occur in vitro or invivo, or can occur partially in vitro and partially in vivo.

[0119] The invention further provides a pharmaceutical compositioncontaining a MEF2 mimetic, which is a peptide or non-peptide moleculethat mimics MEF2 function. The invention further provides apharmaceutical composition containing a p38 mimetic, which is a peptideor non-peptide molecule that mimics p38 function.

[0120] Further provided by the invention is a pharmaceutical compositioncontaining a MEF2 activator and a caspase inhibitor. MEF2 activatorshave been described herein above and include small molecules that resultin increased expression or activity of a MEF2 polypeptide or that mimicMEF2 function. A MEF2 activator can be an organic chemical, drug,nucleic acid molecule, peptide, peptidomimetic, polypeptide or othernaturally or non-naturally occurring organic molecule, and can be, forexample, a MEF2 mimetic. Exemplary MEF2 activators are transcriptionfactors that upregulate MEF2 expression, molecules that compete forbinding to a MEF2 inhibitor such as Cabin1 or histone deacetylase, andkinases that activate MEF2 polypeptides such as p38α.

[0121] In one embodiment, the invention provides a pharmaceuticalcomposition containing a nucleic acid molecule encoding a constitutivelyactive MEF2 polypeptide, and a caspase inhibitor. Constitutively activeMEF2 polypeptides have been described herein and can be prepared bymethods well known in the art. A caspase inhibitor useful in apharmaceutical composition of the invention can be any of the caspaseinhibitors described above or another inhibitor well known in the artand can be, for example, a selective caspase inhibitor.

[0122] The present invention also provides a method of identifying aprotective or differentiation gene, which can be, for example, aneuroprotective gene or a gene that contributes to neuronal or musclecell differentiation. A method of the invention includes the steps ofisolating a first cell population; isolating a second cell population,wherein the second cell population has an altered level or activity of aMEF2 polypeptide as compared to the first cell population; and assayingfor differential gene expression in the first cell population ascompared to the second cell population, whereby a gene differentiallyexpressed in the second cell population as compared to the first cellpopulation is identified as a protective or differentiation gene. In oneembodiment, the first cell population is a progenitor cell population,the second cell population is a neuronal cell population, and thedifferentially expressed gene is a neuronal differentiation gene. In afurther embodiment, the first cell population is a progenitor cellpopulation, the second cell population is a muscle cell population, andthe differentially expressed gene is a muscle differentiation gene. Inyet another embodiment, both cell populations are neuronal cellpopulations, the second cell population has been subject to a neuronalstress as compared to the first cell population, and the differentiallyexpressed gene is a neuroprotective gene.

[0123] It is understood that the term “cell population” can mean asingle cell or a collection of cells. In one embodiment, the first andsecond cell populations each are single cells. Where the cellpopulations are made up of more than a single cell, the cell populationscan be homogeneous or heterogeneous. Furthermore, where the cellpopulations are made up of more than a single cell, it is understoodthat, while the second cell population as a whole has an altered levelor activity of a MEF2 polypeptide as compared to the first cellpopulation as a whole, there may or may not be an altered level oractivity when individual cells from the first and second cellpopulations are compared.

[0124] An altered level of expression or activity of a MEF2 polypeptidecan be achieved, for example, by comparing a particular tissue or cellof interest from a MEF2 knockout or conditional knockout mouse with awild-type littermate. Cells or tissue samples can be obtained, forexample, from the cerebrocortex or hippocampus of the brain or fromcardiac muscle.

[0125] Further provided by the invention is a method of identifying aprotective gene in vitro. The method is practiced by inducing thep38/MEF2 pathway in a cell in vitro to produce a protected cell;stressing the cell; and assaying for differential gene expression in theprotected cell as compared to gene expression in a control cell, wherebya gene differentially expressed in the protected cell as compared to thecontrol cell is identified as a protective gene. In such a method of theinvention, the p38/MEF2 pathway can be induced, for example, byintroducing into the cell a nucleic acid molecule encoding a MEF2polypeptide. The MEF2 polypeptide can be, for example, a human MEF2polypeptide and further can be, if desired, a constitutively active MEF2polypeptide. In one embodiment, a neuroprotective gene is identified byinducing the p38/MEF2 pathway in a neuron. In another embodiment, amuscle protective gene is identified by inducing the p38/MEF2 pathway ina muscle cell. In a method of the invention, the differential geneexpression that identifies the protective gene can be increased ordecreased gene expression.

[0126] The invention additionally provides a method of identifying adifferentiation gene in vitro by inducing the p38/MEF2 pathway in aprogenitor cell in vitro to produce a differentiated cell; and assayingfor differential gene expression in the differentiated cell as comparedto gene expression in a control cell, whereby a gene differentiallyexpressed in the differentiated cell as compared to the control cell isidentified as a differentiation gene. In a method of the invention, thep38/MEF2 pathway can be induced, for example, by introducing into theprogenitor cell a nucleic acid molecule encoding a MEF2 polypeptide. TheMEF2 polypeptide can be, for example, a human MEF2 polypeptide or aconstitutively active MEF2 polypeptide. In one embodiment, thedifferentiated cell is a neuronal cell, and, in a further embodiment,the differentiated cell is a muscle cell. The differential geneexpression which serves to identify the differentiation gene can beincreased or decreased gene expression.

[0127] A variety of means are well known in the art for assaying fordifferential gene expression. Such means include, for example,differential display such as mRNA differential display and differentialdisplay RT-PCR (DDRT-PCR); RNA fingerprinting; subtractive hybridizationapproaches and microarrays such as DNA microarrays. Differential displayanalysis can be used in a method of the invention, for example, asdescribed in Jo et al., Methods Enzymol. 332:233-244 (2001); Staege etal., Immunogenetics 53:105-113 (2001); Fujimoto et al., Hepatol. Res.20:207-215 (2001). In addition, suppressive subtractive hybridizationcan be used to assay for differential gene expression in a method of theinvention, for example, as described in Robert et al., Biol. Reprod.64:1812-1820 (2001). Microarrays such as high-density oligonucleotidearrays and cDNA microarrays also can be useful for assaying fordifferential gene expression in a method of the invention and are wellknown in the art (see, for example, Lee et al., Science 285:1390-1393(1999); Zirlinger et al., Proc. Natl. Acad. Sci., USA 98:5270-5275(2001); Tsunoda et al., Anticancer Res. 21:137-143 (2001) and Khanna etal., Cancer Res. 61:3750-3759 (2001)). One skilled in the artunderstands that these and other methods for assaying for differentialgene expression can be used in a method of the invention.

[0128] The following examples are intended to illustrate but not limitthe present invention.

EXAMPLE I Induction of MEF2 Expression in P19 Cells

[0129] This example demonstrates that retinoic acid induces MEF2 proteinexpression in P19 embryonic carcinoma cells.

[0130] P19 embryonal carcinoma cells terminally differentiate intoneuronal cells after retinoic acid treatment, and the process ofneurogenesis in P19 cells is similar to that of the mammalian centralnervous system (McBurney, Int. J. Dev. Biol. 37:135-140 (1993) and Bainet al., Bioessays 16:343-348 (1994)). Moreover, the apoptotic cell deathobserved in neuronally-differentiating P19 cells parallels that seen inthe fetal brain (Slack et al., J. Cell Biol. 129:779-788 (1995); Mukasaet al., Biochem. Biophys. Res. Commun. 232:192-197 (1997); Blaschke etal., Development 122:1165-1174 (1996); Jacks et al., Nature 359:295-300(1992); and Kuida et al., Nature 384:368-372 (1996)).

[0131] MEF2, especially MEF2C, is expressed during neurogenesis in therodent cerebral cortex (Leifer et al., Proc. Natl. Acad. Sci. USA90:1546-1550 (1993)). To examine if MEF2 proteins are expressed duringneurogenesis of P19 cells, gel shift assays were performed using theMEF2 binding site as a probe. While only very faint binding activity wasdetected in undifferentiated P19 cells, binding activity increased tohigh levels two days after retinoic acid treatment (FIG. 6A, lanes 1 and2). Unlabeled MEF2 oligonucleotide abrogated the binding activity,indicating the specificity of the binding to the MEF2 site (FIG. 6A,lanes 3 and 4). Although anti-MEF2A antibody did not affect theformation of the complex (FIG. 6B, lanes 5 and 6), anti-MEFC andanti-MEF2D antibodies supershifted the bands (FIG. 6B, lanes 7 and 8),indicating the presence of MEF2C and MEF2D proteins in the complex.Immunoblotting revealed that the level of MEF2C and MEF2D proteinsincreased after retinoic acid treatment (FIG. 6C). These resultsindicate that retinoic acid treatment induces MEF2 site-binding activityby MEF2C and MEF2D and an increase in MEF2C and MEF2D protein expressionduring neurogenesis of P19 cells.

[0132] Neuronal differentiation of P19 cells was induced as follows. P19cells were purchased from the ATCC (CRL 1825) and maintained in amodified Eagle's minimum essential medium (MEM; Sigma, St. Louis, Mo.),supplemented with 10% heat inactivated fetal bovine serum (Intergen Co.,Purchase, N.Y.). For neuronal differentiation, 1×10⁶ P19 cells werecultured in a 10 cm diameter tissue culture dish with 300 pM 13-cisretinoic acid (Eastman Kodak, Rochester, N.Y.) for two days. Aftertrypsinization, the cells were again exposed to 300 pM retinoic acid andre-seeded onto a bacterial grade Petri dish to allow the cells toaggregate, thereby facilitating neuronal differentiation. After a oneday incubation, cell aggregates were collected and dissociated withtrypsin-EDTA. The dissociated cells were plated onto a tissue culturechamber slide (Nunc, Rochester, N.Y.). The medium was changed the dayafter plating and every two days thereafter.

[0133] Gel shift assays were performed as follows. Nuclear extracts ofundifferentiated or retinoic acid-treated P19 cells were prepared aspreviously described (Okamoto et al., Brain. Res. Mol. Brain. Res.74:44-54 (1999)). Protein concentrations were measured with a Micro BCAProtein Assay Reagent Kit (Pierce, Rockford, Ill.) using albumin as thestandard. Nuclear extracts (5 μg/20 μl) were preincubated on ice for 10minutes in a solution containing 20 mM Tris (pH 7.6), 10% glycerol, 1 mMdithiothreitol, 80 mM KCl, and 1 μg poly(dI-dC)·(dI-dC). ³²P-end-labeleddouble stranded oligonucleotide representing the MEF2 binding site(TGGGCTATAAATAGCCGC; SEQ ID NO:12) of the brain-specific creatine kinasegene was then added and incubated at room temperature for 20 minutes.The binding mixture was then electrophoresed on a 6% nondenaturingacrylamide gel in 0.25×TBE for 1.5 h at 150 V. For supershift assays,antibodies against MEF2A (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.), MEF2C (Leifer et al., Proc. Natl. Acad. Sci., USA 90:1546-1550(1993)), or MEF2D (provided by Dr. B. Kosofsky, Massachusetts GeneralHospital, Boston) were added to the preincubation binding mixtures.

[0134] Immunoblotting was performed as follows. Whole cell lysates wereprepared in RIPA buffer containing 0.1 mg/ml PMSF and 1 mM sodiumvanadate. Proteins in 50 μg aliquots were separated by SDS-PAGE and thentransferred onto a nitrocellulose membrane (Amersham Life Science,Piscataway, N.J.). Membranes were incubated overnight at 4° C. withprimary antibody to MEF2C (1:1000), MEF2D (1:500), phospho p38 (1:1000,New England Biolabs, Inc., Beverly, Mass.), or p38α (1:1000, Santa CruzBiotech). Horseradish peroxidase-linked anti-rabbit IgG (Vector) wasused as the secondary antibody. Immunoblots were visualized with anenhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech,Piscataway, N.J.).

EXAMPLE II Overexpression of MEF2C in P19 Cells

[0135] These results indicate that stable overexpression of MEF2Ctransforms p19 cells into a mixed neurogenic/myogenic phenotypeexpressing neurofilament as well as the myosin heavy chain.

[0136] P19 cells exposed to DMSO develop into myogenic cells while P19cells exposed to retinoic acid develop along a neurogenic pathway(McBurney, M. W., Int. J. Dev. Biol. 37:135-140 (1993)). To examine thepotential role of MEF2C in the differentiation process, MEF2C wasoverexpressed in stable transformants of undifferentiated P19 cells inthe absence of retinoic acid or DMSO. Undifferentiated P19 cells lackedimmunoreactivity with MEF2C and also lacked neurofilament (FIG. 6E).MEF2C-transfected cells expressed MEF2C protein in the nucleus, asdetermined by specific antibody labeling, and were neuronal in characteras evidenced by labeling with anti-neurofilament. All transfected cells(from over 200 such cells scored) expressed MEF2C, stained withanti-neurofilament, and extended two neuronal-like processes, producinga bipolar appearance (FIG. 6F). None of the MEF2C-transfected cellsstained positively for glial fibrillary acidic protein (GFAP),indicating that they did not manifest an astrocytic phenotype.

[0137] Overexpression of MEF2A has been shown to initiate the myogenicphenotype in the 10T1/2 fibroblast cell line (Kaushal et al., Science266:1236-1240 (1994)) when co-expressed with other factors (MyoD ormyogenin) (Molkentin et al., Cell 83:1125-1136 (1995)). Accordingly, P19cells transfected with MEF2C were assayed for myogenic features bystaining transfected cells with anti-myosin heavy chain antibody. Allcells expressing MEF2C (over 200 counted) were also positive for myosinheavy chain label (FIG. 6G). Taken together, these results indicate thattransfection of undifferentiated P19 cells with MEF2C induces a bipolarcell phenotype that expresses both neuronal (neurofilament) and myogenic(myosin heavy chain) markers.

[0138] MEF2C was overexpressed in P19 cells essentially as follows. Thephosphoglycerate kinase gene promoter-driven expression vector (pGK) waskindly provided by Dr. M. W. McBurney (University of Ottawa, Canada).Human MEF2C cDNA was inserted between the BamHI and XhoI sites of pGK toproduce the expression vector pGK-MEF2C. 2×10⁵ P19 cells were plated ina 6 cm diameter dish 24 h prior to transfection. Subsequently, 25 μg ofthe MEF2C expression vector (pGK-MEF2C) and 1 μg of the neomycinresistance gene expression vector (pSV neo) were co-transfected bycalcium phosphate precipitation. The cells were washed 16 hourspost-transfection and cultured in MEM with 10% serum. After 24 hours,the cells were trypsinized and seeded onto a tissue culture chamberslide. The cells were maintained in 200 μg/ml Geneticin for 5 days toselect the transfected cells.

[0139] Immunocytochemistry was performed as follows. Cultures were fixedwith 3% paraformaldehyde at room temperature for 40 minutes. Afterwashing three times with PBS, cells were permeabilized with 0.3% TritonX-100 for 5 minutes. The free aldehyde groups formed during the fixationwere reduced by incubation with 1 mg/ml sodium borohydride three timesfor 5 minutes each. Cells were then washed three times in PBS. The fixedcells were incubated at 4° C. overnight with primary monoclonalantibodies to microtubule-associated protein2 (MAP2, 1:500; Sigma, cloneHM-2), neurofilament H (1:250; Sternberger Monoclonals Inc.,Lutherville, Md., SMI311), myosin heavy chain (1:250; DevelopmentalStudies Hybridoma Bank, University of Iowa, Iowa, MF20), glialfibrillary acidic protein (GFAP, 1:400; Sigma, clone G-A-5), or rabbitantiserum to MEF2C (1:250) (Leifer et al., supra, 1993). Cells were thenwashed three times in PBS containing 0.2% Tween 20, andrhodamine-conjugated anti-mouse IgG or fluorescein-conjugatedanti-rabbit IgG (each at 1:100; Boehringer Mannheim, Indianapolis, Ind.)was added as the secondary antibody. After a one hour incubation at roomtemperature, the cells were washed again and mounted. For Hu- andnestin-staining, cells were fixed with acid ethanol (95% ethanol: 5%acetic acid) for 30 minutes at room temperature. The fixed cells werethen washed three times with PBS and incubated with a monoclonalantibody to Hu (1:200, gift of Drs. M. F. Marusich and J. A. Weston,University of Oregon, OR) or to nestin (1:20, Developmental StudiesHybridoma Bank, Rat-401). After overnight incubation at 4° C., thesamples were further washed and incubated with anti-mouseimmunoglobulins conjugated to horseradish peroxidase (1:100; DAKO Corp.,Carpinteria, Calif.). A peroxidase reaction was performed using3,3′-diaminobenzidine tetrahydrochloride (Sigma). Stained preparationswere examined under epifluorescence microscopy.

EXAMPLE III Characterization of P19 Clones Stably Expressing a DominantNegative Form of MEF2

[0140] This example describes the characterization of P19 clones inwhich MEF2C function is inhibited.

[0141] The role of endogenous MEF2 proteins in retinoic acid-inducedneuronal differentiation of P19 was analyzed using a dominant negativeform of MEF2. MEF2 proteins are functionally divided into two regions.The N-terminal region (containing the MADS and MEF2 domains) isresponsible for specific DNA binding activity, while the C-terminalregion is necessary for transcriptional activity (Martin et al., Mol.Cell. Biol. 14:1647-1656 (1994), and Molkentin et al., Mol. Cell. Biol.16:2627-2636 (1996)). Since the MADS and MEF2 domains alone lacktranscriptional activity, the N-terminal region of MEF2 acts as adominant negative construct (Martin et al., Mol. Cell. Biol.14:1647-1656 (1994)). Dominant negative MEF2 has been shown to inhibitmyotube formation in myoblastic cell lines (Ornatsky et al., J. Biol.Chem. 272:33271-33278 (1997)).

[0142] Stable transformants of P19 cells were established whichexpressed the dominant negative N-terminus of MEF2C (residues 1 to 105).Three “vector-alone” transfected control clones were designated clones2-1, 2-2 and 2-5; three clones expressing the MEF2 dominant negativewere designated clones 2-7, 2-8 and 2-9. As a further control, twoadditional clones expressing a mutated form of the MEF2 dominantnegative construct were produced and designated clones 2-16 and 2-21.Expression of the MEF2 dominant negative was monitored with gel shiftassays. Binding activity of the MEF2 dominant negative was detected forclones 2-7, 2-8 and 2-9, but not for control clones 2-1, 2-2, 2-5, 2-16,or 2-21 (FIG. 7A and data not shown). All transformants weremorphologically indistinguishable from parent P19 cells.

[0143] The MEF2C dominant negative construct and a control were preparedas follows. Dominant negative MEF2C (amino acids 1-105) tagged with aflag sequence (pcDNAI-MEF2C 1-105 flag) and constitutively active MEF2C(pcDNAI-MEF2C 1-117/VP16) were obtained from Dr. Eric N. Olson(Southwestern Medical Center, Dallas). MEF2C 1-105 flag acts as a MEF2dominant negative by binding to the MEF2 site without producingactivation since it lacks the transactivation domain (Molkentin et al.,Mol. Cell. Biol. 16:2627-2636 (1996)). MEF2C 1-105 flag cDNA was ligatedinto the BamHI/XhoI sites of pGK to produce pGK-DN. A mutation wasengineered in the MEF2 dominant negative by changing the arginineresidue at position 24 to a leucine, and this plasmid was designatedpGK-DNmt. The mutated MEF2 dominant negative was unable to bind to theMEF2 site and therefore served as control to rule out the possibilitythat the MEF2 dominant negative was affecting cell survivalnonspecifically by binding to sites other than MEF2.

[0144] Stable transfection of P19 cells with a dominant negative form ofMEF2 was performed as follows. Using the calcium phosphate precipitationmethod, 2×10⁵ P19 cells were transfected with 24 μg of an emptyexpression vector (pGK), an expression vector encoding a dominantnegative MEF2 (pGK-DN), or a mutated dominant negative MEF2 construct(pGK-DNmt) in addition to the neomycin resistance gene (pSV neo). Thetransfected cells were selected by exposure for 10 days to 200 μg/mlGeneticin. The selection medium was changed every two days. Stableclones expressing the MEF2 dominant negative and the mutated MEF2dominant negative were selected with the reversetranscriptase-polymerase chain reaction.

EXAMPLE IV Inhibition of MEF2 Function Diminishes the Number of Neuronalcells

[0145] This example demonstrates that the number of neuronal cellsformed upon treatment of P19 cells with a neuronal differentiationstimulus is reduced when MEF2C function is inhibited.

[0146] Neuronal differentiation occurs via multiple sequential steps(Stemple and Mahanthappa, Neuron 18:1-4 (1997)). Nerve cellsdifferentiate from unipotent progenitors, which arise from multipotentprecursor cells. The effect of the MEF2 dominant negative on these stepsof neuronal differentiation was monitored by analyzing the appearance ofdifferentiated neurons using antibodies to neuronal markers, such asneurofilament and MAP2. Stable transformants expressing a dominantnegative form of MEF2C which inhibits MEF2 function were treated withretinoic acid to induce neuronal differentiation. After seven days,cells were fixed and stained with anti-MAP2 antibody. Although manyMAP2-positive cells appeared in the control cultures (FIG. 7B), thenumber of MAP2-positive cells was dramatically reduced in the MEF2dominant negative cultures (FIG. 7C). The difference between the numberof MAP2-positive cells in the MEF2 dominant negative cultures and thecontrol cultures was statistically significant (FIG. 7D).

EXAMPLE V Inhibition of MEF2 Function Reduces the Number of Progenitorcells

[0147] This example demonstrates that the number of progenitor cells isreduced by inhibition of MEF2c function with a dominant negativeconstruct.

[0148] The number of precursor cells (multipotent precursor or unipotentprecursor cells) was analyzed following inhibition of MEF2 function. Theappearance of multipotent progenitors (nestin-positive cells) andunipotent precursors (Hu-positive cells) was monitored in control (clone2-1) and MEF2 dominant negative cultures (clone 2-7). Nestin-positiveand Hu-positive cells were counted before and after retinoic acidtreatment for 3.0 days and 3.5 days, respectively. At these time points,multipotent precursor cells (3.0 days) followed by unipotent precursorcells (3.5 days) appear in these cultures prior to the neuronallydifferentiated state (McBurney, M. W., Int. J. Dev. Biol. 37:135-140(1993)). Hu-positive and nestin-positive cells were induced afterretinoic acid treatment in both control (clone 2-1) and MEF2 dominantnegative cultures (clone 2-7; FIG. 7A and C). However, the number ofHu-positive and nestin-positive cells in the MEF2 dominant negativecultures was significantly smaller than than the number of Hu-positiveand nestin-positive cells in control cultures (FIGS. 7B and D). Theseresults indicate that interference with MEF2 activity can reduce thenumber of multipotent precursor and unipotent progenitor cells.

EXAMPLE VI Inhibition of MEF2 Function Enhances Apoptotic Cells DeathDuring Neuronal Differentiation

[0149] This example demonstrates that apoptotic cell death increasesduring neuronal differentiation by inhibition of MEF2C function.

[0150] Apoptotic cell death of differentiating cells is found widely inthe developing fetal brain (Blaschke et al., Development 122:1165-1174(1996)), and apoptosis is also observed during the course of neuronaldifferentiation of P19 cells (Slack et al., J. Cell Biol. 129:779-788(1995), and Mukasa et al., Biochem. Biophys. Res. Commun. 232:192-197(1997)). To examine the number of multipotent and unipotent precursorcells following interference with MEF2 cells, nuclear morphology wasanalyzed with the DNA dye Hoechst 33342 (FIG. 9A) to score the number ofapoptotic cells after three days of retinoic acid treatment, at whichtime multipotent precursors were detected. Prior to the addition ofretinoic acid, fewer than 1% of the cells exhibited apoptotic nuclei incontrol or MEF2 dominant negative cultures. After three days of retinoicacid treatment, a significant number of cells displayed apoptotic nucleiin the controls (transfected with either empty vector or the mutatedform of the MEF2 dominant negative; FIG. 9B and C). However, apoptosiswas increased in the MEF2 dominant negative cultures (FIG. 9B). Thesefindings were confirmed by another apoptosis assay using the TUNELtechnique (FIG. 9D). Additionally, the number of apoptotic cells wasincreased in the MEF2 dominant negative cultures after 3.5 days ofretinoic acid treatment, when unipotent precursor cells predominated.These results indicate that interference with MEF2 activity can increaseapoptotic cell death during neuronal differentiation of P19 cells andcan lead to a reduction in the number of multipotent and unipotentprecursor cells. These results also indicate that MEF2 transcriptionalactivity can be essential for prevention of cell death during neuronaldevelopment.

[0151] Apoptotic assays were performed as follows. Cells were incubatedwith the DNA dye Hoechst 33342 (1 μg/ml) for 5 minutes at 37° C. toobserve nuclear morphology. After washing with PBS, cells were fixedwith acid ethanol (95% ethanol: 5% acetic acid) for 10 minutes at roomtemperature. Samples were then washed with distilled water three timesand mounted. Apoptotic nuclei were counted at 400× magnification. Withinseveral hours of dying by apoptosis, cells underwent secondary necrosis(since they were not phagocytosed in these cultures) and detached fromthe substrate (Bonfoco et al., Proc. Natl. Acad. Sci. USA 92:7162-7166(1995)). Hence, several hours after apoptotic cell death, dead cellswere no longer present to be stained by Hoechst dye. This temporalseparation made it possible to distinguish the number of cells recentlyundergoing apoptosis by using sequential Hoechst staining at differenttime points. Additionally, terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assays wereperformed in a blinded fashion using an In Situ Cell Death Detection Kittagged with tetramethyl-rhodamine (Roche, Nutley, N.J.) or an ApoptosisDetection System Kit tagged with fluorescein (Promega Corporation,Madison, Wis.).

EXAMPLE VII Inhibition of MEF2 Function does not Affect Cell Division ofMultipotent Precursor Cells

[0152] This example demonstrates that MEF2 transcriptional activity hasno significant effect on cell division of multipotent precursor cells.

[0153] Multipotent precursor cells are known to proliferate, leading toan expansion of the cell population that can eventually differentiateinto neurons. Cells transfected with dominant negative MEF2 wereanalyzed for an effect on the proliferation of multipotent precursorcells. Control and MEF2 dominant negative cultures were treated withretinoic acid for 3.0 days, followed by addition of BrdU for detectionof dividing cells. BrdU-positive cells and multipotent precursor cellswere identified by double labeling with anti-BrdU and anti-nestinantibodies, respectively (FIG. 9E). The percentage of cells positive forboth BrdU and nestin (proliferating, multipotent precursor cells) wassimilar in the control and MEF2 dominant negative cultures (FIG. 9F).These results indicate that MEF2 transcriptional activity has nosignificant effect on cell division of multipotent precursor cells.

[0154] Cell proliferation assays were performed as follows. To labelproliferating cells, bromodeoxyuridine (BrdU; Amersham Life Science) wasadded to cultures at a dilution of 1:1000 for two hours at 37° C. Afterwashing with PBS, cells were treated with acid ethanol (95% ethanol: 5%acetic acid) for 30 minutes at room temperature and cellular DNA wasdenatured with 2N HCl. After an additional wash in PBS, cells wereincubated at 4° C. overnight in rat monoclonal anti-BrdU antibody (1:10;Harlan Sera-Lab Limited, Indianapolis, Ind.) and mouse monoclonalanti-nestin antibody (11 μg/ml). After three washes in PBS, secondaryantibodies were added: Rhodamine Red-X-conjugated anti-rat IgG (1:200)and Biotin-SP-conjugated anti-mouse IgG (1:50; Jackson ImmunoResearchLaboratories, Inc.; Westgrove, Pa.) followed by streptavidin-fluorescein(1:25; Amersham Life Science Inc.). Cells were examined underepifluorescence microscopy.

EXAMPLE VIII Inhibition of P38 Map Kinase Increases Apoptosis DuringNeuronal Differentiation

[0155] This example demonstrates that the p38α/MEF2 cascade plays a rolein preventing apoptotic cell death during neuronal differentiation.

[0156] Interference with MEF2 function enhanced apoptosis indifferentiating P19 cells, indicating that MEF2 transcriptional activitywas necessary for prevention of cell death during neuronal development.Two members of the p38 MAP kinase family, p38α and p38β₂, are known toactivate MEF2 via phosphorylation of Ser/Thr residues (Han et al.,Nature 386:296-299 (1997); Zhao et al., Mol. Cell. Biol. 19:21-30(1999); and Yang et al., Mol. Cell. Biol. 19:4028-4038 (1999)).Moreover, the p38 MAP kinase family can play a role in cell survival inseveral cell types (New and Han, Trends Cardiovasc. Med. 8:220-229(1998)).

[0157] The p38/MEF2 pathway was examined for a role in preventingapoptosis in differentiating P19 cells. Activation of p38 family memberswas examined by immunoblotting with an anti-phospho-pan p38 antibody,which recognizes all activated/phosphorylated p38 MAP kinases. One bandwas strongly induced after retinoic acid treatment, and the mobility ofthis band was the same as p38α (FIG. 10A). Total (phosphorylated andunphosphorylated) p38α protein appeared to be present at similar levelsbefore and after stimulation with retinoic acid (FIG. 10A, right-handlanes). In contrast, p38β₂ was undetectable with two differentantibodies, although these antibodies clearly reacted with recombinantp38β₂ protein under the same conditions in control studies. Theseresults indicate that p38α is activated/phosphorylated during theinduction of neurogenesis.

[0158] Transfection of dominant negative p38α, but not dominant negativep38β₂, enhanced apoptotic cell death in differentiating cells.Furthermore, co-expression of constitutively active MEF2C significantlyrescued these differentiating cells from apoptosis (FIG. 10B). Thesefindings indicate that the p38α/MEF2 cascade plays a role in preventingapoptotic cell death during neuronal differentiation.

[0159] Transient transfection during differentiation of P19 cells wasperformed as follows. P19 cells (2×10⁵) were seeded onto 6-well tissueculture plates and treated with 300 pM retinoic acid to induce neuronaldifferentiation. One day later, cells were transfected with 0.83 μg of ap38 dominant negative construct or vector alone [pcDNA3-p38α(AF),pcDNA3-p38β₂ (AF) or pcDNA3], 0.83 μg of a constitutively active MEF2Cconstruct or vector alone (pcDNAI-MEF2C 1-117/VP16 or pcDNAI-Amp), plus0.33 μg of a Green Fluorescent Protein (GFP) construct to identifytransfected cells (pEGFP-N1). A lipid based transfection system wasutilized (6 ml of TransFast, Promega). The next day cells weretransferred to 3.5-cm bacterial dishes and treated with an additional300 pM retinoic acid. TUNEL assays were performed on day three afterinitiating retinoic acid differentiation. Over 1200 GFP-positive cellswere scored for apoptosis from each culture plate under epifluorescencemicroscopy, and each experiment was replicated on three separate days.

EXAMPLE IX Expression of Constitutively Active MEF2 in UndifferentiatedES Cells

[0160] A. Expression of Constitutively Active MEF2

[0161] Murine embryonic stem cells (ES cell line D3) were plated at a1:5 dilution in ES Cell Passage Medium (DMEM with 20% fetal bovineserum, 2250 mg/L glucose, MEM non-essential amino acids, 1 mM sodiumpyruvate and penicillin-streptomycin) in gelatin-coated 24 well plates.One day later, ES cells were incubated for five hours with a mixture of1.25 μg of vector alone (pCDNA1) or pCDNA expression vector encodingconstitutively active MEF2 (pCDNA MEF2C/VP16), 0.25 μg expression vectorencoding green fluorescence protein (GFP; pEGFPN1; Clontech), and 3 μlof LipofectAMINE 2000 (Promega). Transfected cells were identified byexpression of GFP. Cells were washed twice with Neuron Induction Medium(DMEM with 10% bovine calf serum, 4500 mg/L glucose, 10% F-12 growthsupplement, 1 mM glutamine, 25 mM HEPES (pH 7.0) andpenicillin-streptomycin). Further incubation for one day in NeuronInduction Medium resulted in differentiation of ES cells to neurons asindicated by the presence of neuron-specific markers (see below).

[0162] B. Characterization of Differentiated ES Cells

[0163] Cells were analyzed for expression of the neuron-specific marker,neurofilament H essentially as follows. Cells were trypsinized andtransferred into a four-well chamber slide (Nunc) coated with lamininand poly-L-lysine. Two days later, cells were fixed with 3%paraformaldehyde and permeabilized with 0.3% Triton-X 100. Expression ofneurofilament H was determined with monoclonal antibody toanti-neurofilament H monoclonal antibody SMI311 (SternbergerMonoclonals) and detected with anti-mouse IgG conjugated to rhodamineRed-X (Jackson ImmunoResearch). Red (neurofilament H-positive,transfected (GFP-positive) cells were scored using epifluorescencemicroscopy. The results showed that eighty-three percent of transfectedcells displayed expression of neurofilament H, indicating a high degreeof efficiency in conversion of murine ES cells transfected withconstitutively active MEF2C to neurons. Similar results were obtainedwith ES cells transfected with constitutively active MEF2A/VP16.

[0164] These results demonstrate that overexpression of constitutivelyactive MEF2 (MEF2/VP16) induces neurogenesis in undifferentiated EScells.

[0165] All journal article, reference, and patent citations providedabove, in parentheses or otherwise, whether previously stated or not,are incorporated herein by reference.

[0166] Although the invention has been described with reference to theexamples above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

We claim:
 1. A method of differentiating progenitor cells, comprisingthe steps of: (a) contacting said progenitor cells with adifferentiating agent; and (b) introducing into said progenitor cells anucleic acid molecule encoding a MEF2 polypeptide or an active fragmentthereof, thereby differentiating said progenitor cells to produce a cellpopulation containing protected neuronal cells.
 2. The method of claim1, wherein said MEF2 polypeptide is human MEF2C, or an active fragmentthereof.
 3. The method of claim 1, wherein said MEF2 polypeptide isconstitutively active.
 4. The method of claim 3, wherein saidconstitutively active MEF2 polypeptide is a MEF2/VP16 fusion protein. 5.The method of claim 3, wherein said constitutively active MEF2polypeptide contains one or more serine/threonine to asparticacid/glutamic acid substitutions in the MEF2 transactivation domain. 6.The method of claim 1 or claim 3, further comprising inhibiting caspaseactivity in said progenitor cells.
 7. The method of claim 1, whereinsaid progenitor cells are human stem cells.
 8. The method of claim 1,wherein said progenitor cells are embryonic stem cells.
 9. The method ofclaim 8, wherein said embryonic stem cells are human embryonic stemcells.
 10. The method of claim 1, wherein said progenitor cells arehematopoietic progenitor cells.
 11. The method of claim 10, wherein saidhematopoietic progenitor cells are human hematopoietic progenitor cells.12. The method of claim 1, further comprising selecting CD133-positivehuman progenitor cells.
 13. The method of claim 1, further comprisingselecting CD133-positive/CD34-positive human progenitor cells.
 14. Themethod of claim 1, further comprising selectingCD133-positive/CD34-negative human progenitor cells.
 15. The method ofclaim 1, further comprising selectingCD133-positive/CD34-negative/CD45-negative human progenitor cells. 16.The method of claim 1, further comprising selectingCD34-negative/CD38-negative/Lin-negative human progenitor cells.
 17. Themethod of claim 1, further comprising selectingCD34-positive/CD38-negative/Lin-negative/Thy-1-negative human progenitorcells.
 18. The method of claim 1, wherein said differentiating agent isretinoic acid.
 19. The method of claim 1, wherein said differentiatingagent is selected from the group consisting of neurotrophic factor 3,epidermal growth factor, insulin-like growth factor 1 and aplatelet-derived growth factor.
 20. The method of claim 1, wherein saidpopulation containing protected neuronal cells comprises at least 50%neuronal cells.
 21. The method of claim 1, further comprising the stepof (c) transplanting cells comprising a nucleic acid molecule encoding aMEF2 polypeptide or an active fragment thereof into a patient to producea cell population containing protected neuronal cells in said patient.22. An isolated stem cell, comprising an exogenous nucleic acid moleculeencoding a MEF2 polypeptide or an active fragment thereof.
 23. Theisolated stem cell of claim 22, comprising a nucleic acid moleculeencoding a MEF2 polypeptide, or active fragment thereof, operativelylinked to a heterologous regulatory element.
 24. The isolated stem cellof claim 22, wherein said MEF2 polypeptide is a human MEF2 polypeptide.25. The isolated stem cell of claim 22, wherein said MEF2 polypeptide isa MEF2C polypeptide.
 26. The isolated stem cell of claim 22, whereinsaid MEF2 polypeptide is constitutively active.
 27. The isolated stemcell of claim 26, wherein said MEF2 polypeptide is a constitutivelyactive MEF2C polypeptide.
 28. The isolated stem cell of claim 26,wherein said constitutively active MEF2 polypeptide is a MEF2/VP16fusion protein.
 29. The isolated stem cell of claim 26, wherein saidconstitutively active MEF2 polypeptide contains one or moreserine/threonine to aspartic acid/glutamic acid substitutions in theMEF2 transactivation domain.
 30. The isolated stem cell of claim 22,which is a human stem cell.
 31. The isolated stem cell of claim 22,which is an embryonic stem cell.
 32. The isolated embryonic stem cell ofclaim 31, which is a human embryonic stem cell.
 33. The isolated humanstem cell of claim 30, wherein said MEF2 polypeptide is human MEF2C. 34.The isolated human stem cell of claim 30, wherein said MEF2 polypeptideis constitutively active.
 35. An isolated hematopoietic stem cell,comprising an exogenous nucleic acid molecule encoding a MEF2polypeptide or an active fragment thereof.
 36. The isolatedhematopoietic stem cell of claim 35, comprising a nucleic acid moleculeencoding a MEF2 polypeptide, or active fragment thereof, operativelylinked to a heterologous regulatory element.
 37. The isolatedhematopoietic stem cell of claim 36, which is a human hematopoietic stemcell.
 38. A method of identifying a protective or differentiation gene,comprising: (a) isolating a first cell population; (b) isolating asecond cell population, wherein said second cell population has analtered level or activity of a MEF2 polypeptide as compared to saidfirst cell population; and (c) assaying for differential gene expressionin said first cell population as compared to said second cellpopulation, whereby a gene differentially expressed in said second cellpopulation as compared to said first cell population is identified as aprotective or differentiation gene.
 39. The method of claim 38, whereinsaid first cell population is a progenitor cell population, said secondcell population is a neuronal cell population, and said differentiallyexpressed gene is a neuronal differentiation gene.
 40. The method ofclaim 38, wherein said first cell population is a progenitor cellpopulation, said second cell population is a muscle cell population, andsaid differentially expressed gene is a muscle differentiation gene. 41.The method of claim 38, wherein said first and second cell populationsare neuronal cell populations, said second cell population has beensubject to a neuronal stress as compared to said first cell population,and said differentially expressed gene is a neuroprotective gene.
 42. Amethod of identifying a protective gene in vitro, comprising the stepsof: (a) inducing the p38/MEF2 pathway in a cell in vitro to produce aprotected cell; (b) stressing said cell; and (c) assaying fordifferential gene expression in said protected cell as compared to geneexpression in a control cell, whereby a gene differentially expressed insaid protected cell as compared to said control cell is identified as aprotective gene.
 43. The method of claim 42, wherein step (a) comprisesintroducing into said cell a nucleic acid molecule encoding a MEF2polypeptide.
 44. The method of claim 42, wherein said MEF2 polypeptideis a human MEF2 polypeptide.
 45. The method of claim 42, wherein saidMEF2 polypeptide is a constitutively active MEF2 polypeptide.
 46. Themethod of claim 42, wherein said cell is a neuron.
 47. The method ofclaim 42, wherein said cell is a muscle cell.
 48. The method of claim42, wherein said differential gene expression is increased geneexpression.
 49. The method of claim 42, wherein said differential geneexpression is decreased gene expression.
 50. A method of identifying adifferentiation gene in vitro, comprising the steps of: (a) inducing thep38/MEF2 pathway in a progenitor cell in vitro to produce adifferentiated cell; and (b) assaying for differential gene expressionin said differentiated cell as compared to gene expression in a controlcell, whereby a gene differentially expressed in said differentiatedcell as compared to said control cell is identified as a differentiationgene.
 51. The method of claim 50, wherein step (a) comprises introducinginto said progenitor cell a nucleic acid molecule encoding a MEF2polypeptide.
 52. The method of claim 50, wherein said MEF2 polypeptideis a human MEF2 polypeptide.
 53. The method of claim 50, wherein saidMEF2 polypeptide is a constitutively active MEF2 polypeptide.
 54. Themethod of claim 50, wherein said differentiated cell is a neuronal cell.55. The method of claim 50, wherein said differentiated cell is a musclecell.
 56. The method of claim 50, wherein said differential geneexpression is increased gene expression.
 57. The method of claim 50,wherein said differential gene expression is decreased gene expression.